Conjugated peptide nucleic acids having enhanced cellular uptake

ABSTRACT

Peptide nucleic acids conjugated to lipophilic groups and incorporated into liposomes exhibit enhanced cellular uptake and distribution. Cellular uptake and distribution of peptide nucleic acids also increases with the introduction of an amino acid side chain into the backbone of peptide nucleic acids. Methods of modulating cellular uptake and methods for treating animals are provided. The peptide nucleic acids of the invention comprise naturally-occurring nucleobases and non-naturally-occurring nucleobases attached to a polyamide backbone.

FIELD OF THE INVENTION

[0001] The present invention is directed to compositions comprising apeptide nucleic acid (PNA) which is conjugated to a lipophilic group andincorporated into liposomes. The PNA is composed of naturally-occurringnucleobases or non-naturally-occurring nucleobases which are covalentlybound to a polyamide backbone. The PNA compositions of the presentinvention may further comprise a PNA modified by an amino acid sidechain. The PNA compositions of the present invention exhibit enhancedcellular uptake and distribution. PNA compositions which wereincorporated into liposomes demonstrated increased cellular uptake andmore diffuse distribution than PNA compositions without liposomes.

BACKGROUND OF THE INVENTION

[0002] The function of a gene starts by transcription of its informationto a messenger RNA (mRNA). By interacting with the ribosomal complex,mRNA directs synthesis of proteins. This protein synthesis process isknown as translation. Translation requires the presence of variouscofactors, building blocks, amino acids and transfer RNAs (tRNAs), allof which are present in normal cells.

[0003] Most conventional drugs exert their effect by interacting withand modulating one or more targeted endogenous proteins, e.g., enzymes.Typically, however, such drugs are not specific for targeted proteinsbut interact with other proteins as well. Thus, use of a relativelylarge dose of drug is necessary to effectively modulate the action of aparticular protein. If the modulation of a protein activity could beachieved by interaction with or inactivation of mRNA, a dramaticreduction in the amount of drug necessary and in the side-effects of thedrug could be achieved. Further reductions in the amount of drugnecessary and the side-effects could be obtained if such interaction issite-specific. Since a functioning gene continually produces mRNA, itwould be even more advantageous if gene transcription could be arrestedin its entirety. Oligonucleotides and their analogs have been developedand used as diagnostics, therapeutics and research reagents. One exampleof a modification to oligonucleotides is labeling with non-isotopiclabels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, orother reporter molecules. Other modifications have been made to theribose phosphate backbone to increase the resistance to nucleases. Thesemodifications include use of linkages such as methyl phosphonates,phosphorothioates and phosphorodithioates, and 2′-O-methyl ribose sugarmoieties. Other oligonucleotide modifications include those made tomodulate uptake and cellular distribution. Phosphorothioateoligonucleotides are presently being used as antisense agents in humanclinical trials for the treatment of various disease states. Althoughsome improvements in diagnostic and therapeutic uses have been realizedwith these oligonucleotide modifications, there exists an ongoing demandfor improved oligonucleotide analogs.

[0004] There are several known nucleic acid analogs having nucleobasesbound to backbones other than the naturally-occurring ribonucleic acidsor deoxyribonucleic acids. These nucleic acid analogs have the abilityto bind to nucleic acids with complementary nucleobase sequences. Amongthese, the peptide nucleic acids (PNAs), as described, for example, inWO 92/20702, have been shown to be useful as therapeutic and diagnosticreagents. This may be due to their generally higher affinity forcomplementary nucleobase sequence than the corresponding wild-typenucleic acids.

[0005] PNAs are useful surrogates for oligonucleotides in binding to DNAand RNA. Egholm et al., Nature, 1993, 365, 566, and references citedtherein. The current literature reflects the various applications ofPNAs. Hyrup et al., Bioorganic & Med. Chem., 1996, 4, 5; and Nielsen,Perspectives Drug Disc. Des., 1996, 4, 76.

[0006] PNAs are compounds that are analogous to oligonucleotides, butdiffer in composition. In PNAs, the deoxyribose backbone ofoligonucleotide is replaced by a peptide backbone. Each subunit of thepeptide backbone is attached to a naturally-occurring ornon-naturally-occurring nucleobase. One such peptide backbone isconstructed of repeating units of N-(2-aminoethyl)glycine linked throughamide bonds. The synthesis of PNAs via preformed monomers was previouslydescribed in WO 92/20702 and WO 92/20703, the contents of which areherein incorporated by reference. More recent advances in the structureand synthesis of PNAs are illustrated in WO 93/12129 and U.S. Pat. No.5,539,082, issued Jul. 23, 1996, the contents of both being hereinincorporated by reference. Further, the literature is replete withpublications describing synthetic procedures, biological properties anduses of PNAs. For example, PNAs possess the ability to effect stranddisplacement of double-stranded DNA. Patel. Nature, 1993, 365, 490.Improved synthetic procedures for PNAs have also been described. Nielsenet al., Science, 1991, 254, 1497; and Egholm, J. Am. Chem. Soc., 1992,114, 1895. PNAs form duplexes and triplexes with complementary DNA orRNA. Knudson et al., Nucleic Acids Research, 1996, 24, 494; Nielsen etal., J. Am. Chem. Soc., 1996, 118, 2287; Egholm et al., Science, 1991,254, 1497; Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895; and Egholmet al., J. Am. Chem. Soc., 1992, 114, 9677.

[0007] PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNAduplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound tighterthan corresponding DNA/DNA or DNA/RNA duplexes as evidenced by theirhigher melting temperatures (T_(m)). This high thermal stability ofPNA/DNA(RNA) duplexes has been attributed to the neutrality of the PNAbackbone, which results elimination of charge repulsion that is presentin DNA/DNA or RNA/RNA duplexes. Another advantage of PNA/DNA(RNA)duplexes is that T_(m) is practically independent of salt concentration.DNA/DNA duplexes, on the other hand, are highly dependent on the ionicstrength.

[0008] Triplex formation by oligonucleotides has been an area of intenseinvestigation since sequence-specific cleavage of double-strandeddeoxyribonucleic acid (DNA) was demonstrated. Moser et al., Science,1987, 238, 645. The potential use of triplex-forming oligonucleotides ingene therapy, diagnostic probing, and other biomedical applications hasgenerated considerable interest Uhlmann et al., Chemical Reviews, 1990,90, 543. Pyrimidine oligonucleotides have been shown to form triplehelix structures through binding to homopurine targets indouble-stranded DNA. In these structures the new pyrimidine strand isoriented parallel to the purine Watson-Crick strand in the major grooveof the DNA and binds through sequence-specific Hoogsteen hydrogenbonding. The sequence specificity is derived from thymine recognizingadenine (T:A-T) and protonated cytosine recognizing guanine (C⁺:G-C).Best et al., J. Am. Chem. Soc., 1995, 117, 1187. In a less well-studiedtriplex motif, purine-rich oligonucleotides bind to purine targets ofdouble-stranded DNA. The orientation of the third strand in this motifis anti-parallel to the purine Watson-Crick strand, and the specificityis derived from guanine recognizing guanine (G:G-C) and thymine oradenine recognizing adenine (A:A-T or T:A-T). Greenberg et al., J. Am.Chem. Soc., 1995, 117, 5016.

[0009] Homopyrimidine PNAs have been shown to bind complementary DNA orRNA forming (PNA)₂/DNA(RNA) triplexes of high thermal stability. Egholmet al., Science, 1991, 254, 1497; Egholm et al., J. Am. Chem. Soc.,1992, 114, 1895; Egholm et al., J. Am. Chem. Soc., 1992, 114, 9677. Theformation of triplexes involving two PNA strands and one nucleotidestrand has been reported in U.S. patent application Ser. No. 08/088,661,filed Jul. 2, 1993, the contents of which are incorporated herein byreference. The formation of triplexes in which the Hoogsteen strand isparallel to the DNA purine target strand is preferred to formation ofanti-parallel complexes. This allows for the use of bis-PNAs to obtaintriple helix structures with increased pH-independent thermal stabilityusing pseudoisocytosine instead of cytosine in the Hoogsteen strand.Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895. Further, see WO96/02558, the contents of which are incorporated herein by reference.

[0010] Peptide nucleic acids have been shown to have higher bindingaffinities (as determined by their Tm's) for both DNA and RNA than thatof DNA or RNA to either DNA or RNA. This increase in binding affinitymakes these peptide nucleic acid oligomers especially useful asmolecular probes and diagnostic agents for nucleic acid species.

[0011] In addition to increased affinity, PNAs have increasedspecificity for DNA binding. Thus, a PNA/DNA duplex mismatch show 8 to20° C. drop in the T_(m) relative to the DNA/DNA duplex. This decreasein T_(m) is not observed with the corresponding DNA/DNA duplex mismatch.Egholm et al., Nature 1993, 365, 566.

[0012] A further advantage of PNAs, compared to oligonucleotides, isthat the polyamide backbone of PNAs is resistant to degradation byenzymes.

[0013] Considerable research is being directed to the application ofoligonucleotides and oligonucleotide analogs that bind to complementaryDNA and RNA strands for use as diagnostics, research reagents andpotential therapeutics. For many applications, the oligonucleotides andoligonucleotide analogs must be transported across cell membranes ortaken up by cells to express their activity.

[0014] PCT/EP/01219 describes novel PNAs which bind to complementary DNAand RNA more tightly than the corresponding DNA. It is desirable toappend groups to these PNAs which will modulate their activity, modifytheir membrane permeability or increase their cellular uptake property.One method for increasing amount of cellular uptake property of PNAs isto attach a lipophilic group. U.S. application Ser. No. 117,363, filedSep. 3, 1993, describes several alkylamino functionalities and their usein the attachment of such pendant groups to oligonucleosides.

[0015] U.S. application Ser. No. 07/943,516, filed Sep. 11, 1992, andits corresponding published PCT application WO 94/06815, describe othernovel amine-containing compounds and their incorporation intooligonucleotides for, inter alia, the purposes of enhancing cellularuptake, increasing lipophilicity, causing greater cellular retention andincreasing the distribution of the compound within the cell.

[0016] U.S. application Ser. No. 08/116,801, filed Sep. 3, 1993,describes nucleosides and oligonucleosides derivatized to include athiolalkyl functionality, through which pendant groups are attached.

[0017] Recently, liposomal drug-delivery systems incorporating variousbiomolecules and drugs have been studied and found to exhibit reducedtoxicities and increased efficacy due to enhanced cellular uptake anddistribution. Chonn and Cullis, Current Opinion in Biotechnology, 1995,6, 698; Mannino et al., Biotechniques, 1988, 6, 682; Blume and Cevc,Biochem et Biophys. Acta, 1990, 1029, 91; and Lappalainen et al.,Antiviral Res., 1994, 23, 119. Liposomes are microscopic spherescomposed of an aqueous core and a lipid bilayer enveloping the core.Procedures for preparation of liposomes are available in the literature.G. Gregoridadis in “Liposome Technology,” volume 2, G. Gregoridadis(ed.), CRC Press, 1993, p. 1; Watwe and Bellare, Curr. Sci., 1995, 68,715. Several liposomal drugs are currently on the market or underdevelopment. Chonn and Cullis, Current Opinion in Biotechnology, 1995,6, 698.

[0018] WO 96/10391, published Apr. 11, 1995, describes polyethyleneglycol-modified ceramide lipids which are used to form liposomes, andthe use of these liposomes as drug-delivery vehicles.

[0019] WO 96/24334, published Aug. 15, 1996, describes lipid constructshaving an aminomannose-derivatized cholesterol moiety for the deliveryof drugs to the cytoplasm of cells, particularly to vascular smoothmuscle tissues.

[0020] WO 96/40627, published Dec. 19, 1996, describes cationiclipid-containing liposome formulations which are useful in the deliveryof biomolecules such as oligonucleotides, nucleic acids, peptides andother agents.

[0021] Despite recent advances, there remains a need for stablecompositions with enhanced cellular uptake and distribution.

SUMMARY OF THE INVENTION

[0022] The present invention provides peptide nucleic acids (PNAs)conjugated to a lipophilic group and having a modified backbone whereinan amino acid side chain is attached to the backbone. The presentinvention also provides liposomal compositions comprising a peptidenucleic acid (PNA) conjugated to a lipophilic group which isincorporated into liposomes. The PNAs of the present invention comprisenucleobases covalently bound to a polyamide backbone. Representativenucleobases include the four major naturally-occurring DNA nucleobases(i.e., thymine, cytosine, adenine and guanine), othernaturally-occurring nucleobases (e.g. inosine, uracil, 5-methylcytosine,thiouracil and 2,6diaminopurine) and artificial nucleobases (e.g.,bromothymine, azaadenines and azaguanines). These nucleobases areattached to a polyamide backbone through, a suitable linker.

[0023] Preferred peptide nucleic acids of the invention have the generalformula (I):

[0024] wherein:

[0025] each L is, independently, a naturally-occurring nucleobase or anon-naturally-occurring nucleobase;

[0026] each R^(T) is hydrogen or the side chain of a naturally-occurringor non-naturally-occurring amino acid, at least one R^(T) being the sidechain of an amino acid;

[0027] R^(h) is OH, NH₂, or NHLysNH_(2;)

[0028] each of R^(i) and R^(j) is, independently, a lipophilic group oran amino acid labeled with a fluorescent group; or R^(i) and R^(j),together, are a lipophilic group;

[0029] n is an integer from 1 to 30.

[0030] PNAs having formula (I) wherein R^(i) is D-lysine labeled with afluorescent group and R^(J) is an adamantoyl group are preferred. Evenmore preferred are PNAs of formula (I) wherein R^(i) is D-lysine labeledwith fluorescein and R^(j) is an adamantoyl group. Also preferred arePNAs having formula (I) wherein R^(i) and R^(j), together, are anadamantoyl group. Further preferred are PNAs of formula (I) wherein atleast one of said R^(T) is the side chain of D-lysine.

[0031] Preferably, the carbon atom to which substituent R^(T) isattached is stereochemically enriched. Hereinafter, “stereochemicallyenriched” means that one stereoisomer predominates over the otherstereoisomer in a sufficient amount as to provide a beneficial effect.Preferably, one stereoisomer predominates by more than 50%. Morepreferably, one stereoisomer predominates by more than 80%. Even morepreferably, one stereoisomer predominates by more than 90%. Still morepreferably, one stereoisomer predominates by more than 95%. Even morepreferably, one stereoisomer predominates by more than 99%. Still evenmore preferably, one stereoisomer is present substantiallyquantitatively.

[0032] The present invention also provides liposomal compositionscomprising a peptide nucleic acid incorporated in a liposome, saidpeptide nucleic acid having formula (I) wherein:

[0033] each L is, independently, a naturally-occurring nucleobase or anon-naturally-occurring nucleobase;

[0034] each R^(T) is hydrogen or the side chain of a naturally-occurringor non-naturally-occurring amino acid;

[0035] R^(h) is OH, NH₂, or NHLysNH_(2;)

[0036] each of R^(i) and R^(j) is, independently, a lipophilic group oran amino acid labeled with a fluorescent group; or R^(i) and R^(j),together, are a lipophilic group;

[0037] n is an integer from 1 to 30.

[0038] PNAs having formula (I) wherein R^(i) is D-lysine labeled with afluorescent group and R^(j) is an adamantoyl group are preferred. Evenmore preferred are PNAs of formula (I) wherein R^(i) is D-lysine labeledwith fluorescein and R^(j) is an adamantoyl group. Also preferred arePNAs having formula (I) wherein R^(i) and R^(j), together, are anadamantoyl group. Further preferred are PNAs of formula (I) wherein atleast one of said R^(T) is the side chain of D-lysine.

[0039] Preferably, the carbon atom to which substituent R^(T) isattached is stereochemically enriched.

[0040] The PNAs of the present invention are synthesized by adaptationof standard peptide synthesis procedures, either in solution or on asolid phase.

[0041] The present invention further provides methods for enhancing thecellular uptake and distribution of peptide nucleic acids byincorporation of amino acid side chains into PNA backbones, conjugatinglipophilic groups with PNAs and introducing PNAs into liposomes.

BRIEF DESCRIPTION OF THE DRAWING

[0042]FIG. 1 shows structures of some lipophilic groups.

DETAILED DESCRIPTION OF THE INVENTION

[0043] In accordance with the present invention, peptide nucleic acidsand liposomal compositions exhibiting enhanced cellular uptake anddistribution are provided. The peptide nucleic acids (PNAs) of theinvention are assembled from a plurality of nucleobases which areattached to a polyamide backbone by a suitable linker. In one preferredembodiment of the present invention, the PNAs are conjugated to alipophilic group. As used herein, “conjugating” refers to attaching alipophilic group to a PNA of the invention. In another preferredembodiment, the polyamide backbone of PNAs of the invention isderivatized. As used herein, “derivatizing” refers to modifying thebackbone of a PNA by attaching the side chain of at least onenaturally-occurring or non-naturally-occurring amino acid to thepolyamide backbone. The liposomal compositions of the present inventioncomprise peptide nucleic acids of the invention that are incorporatedinto liposomes. Thus, the liposomal compositions of the presentinvention comprise PNAs which are encapsulated by liposomes. The PNAsand liposomal compositions of the present invention exhibit enhancedcellular uptake and distribution.

[0044] The PNAs of the present invention have the formula (I) whereinnucleobase L is a naturally-occurring nucleobase attached at theposition found in nature, i.e., position 9 for adenine or guanine, andposition 1 for thymine or cytosine, a non-naturally-occurring nucleobase(nucleobase analog) or a nucleobase-binding moiety. Representativenucleobases include the four major naturally-occurring DNA nucleobases(i.e., thymine, cytosine, adenine and guanine), othernaturally-occurring nucleobases (e.g. inosine, uracil, 5-methylcytosine,thiouracil and 2,6-diaminopurine) and artificial nucleobases (e.g.,bromothymine, azaadenines and azaguanines). These nucleobases areattached to a polyamide backbone through a suitable linker.

[0045] The PNAs of formula (I) include one or more amino acid moietieswithin their structure. These amino acids may be naturally-occurring ornon-naturally-occurring. Naturally-occurring amino acids include α-aminoacids where the chiral center has a D-configuration. Suchnaturally-occurring amino-acids may be either essential or non-essentialamino acids. Non-naturally-occurring amino acids used in the PNAs of thepresent invention of formula (I) include α-amino acids with chiralcenters bearing an L-configuration. Non-naturally-occurring amino acidsalso include amino acids bearing unusual side chains that do not existin nature and are prepared synthetically, such as halo- andcyano-substituted benzyl, tetrahydroisoquinolylmethyl, cyclohexylmethyl,and pyridylmethyl. Other synthetic amino-acids include β-amino acids.

[0046] The amino acids may be introduced into the PNAs of formula (I)either as part of the monomer used or at the terminal ends of the PNA.Any of the abovementioned amino acids could be incorporated into themonomeric building blocks used in PNA synthesis. Preferably the aminoacid used is glycine, where R^(T) is H. R^(T) can also be methyl, ethyl,benzyl, isopropyl, p-hydroxybenzyl, halobenzyl, carboxymethyl,tetrahydroisoquinolinylmethyl, or aminohexanoyl. Amino acids may also beattached at the C-terminus of PNAs such that the terminal R^(h)—CO—group represents an amino acyl group derived from any naturally- ornon-naturally-occurring amino acid, α- or β-amino acid, and with a D- orL-configuration at the α-chiral center. Preferably the C-terminal aminoacid is lysine. Amino acids may also be incorporated at the N-terminalend of the PNA of structure (I) where each of R^(i) and R^(j) may,independently, be an amino acyl group derived from any naturally- ornon-naturally-occurring amino acid, α- or β-amino acid, and with a D- orL-configuration at the α-chiral center. Preferably the N-terminal aminoacid is lysine.

[0047] Lipophilic groups attached to PNA's of formula (I) of the presentinvention, include natural and synthetic fatty acids, fatty alcoholderivatives and diacylglycerol derivatives such as adipic acid, palmiticacid, decanoic acid, octadecanoic acid, oleic acid, elaidic acid,linoleic acid, bile acids, heptylsuccinic acid, palmitylsuccinic acid,polyglycolic acid, dioctadecylglycerol phosphatidic acid,dioleoylglycerol phosphatidic acid, adamantoyl, octadecyloxycarbonyl,and decalinoyl. These lipophilic groups may be attached at any suitablelocation in the PNA molecule of formula (I). Preferably, the lipophilicgroup is attached to the N-terminus of the PNA of the invention whereineach of R^(i) and R^(j) may, independently, be a lipophilic group. Morepreferably, R^(i) and R^(j), together, are an adamantoyl group.

[0048] The PNA of the present invention have the formula (I) whereinlabels, such as fluorescent groups, are incorporated so as to allow aconvenient means by which to detect the PNA. Fluorescent groups include,but are not limited to, dyes such as fluorescein, rhodamine, pyrenyl,cyanine dyes, Cy5™ (Biological Detection Systems, Inc., Pittsburgh,Pa.), and derivatives of such dyes. These may be incorporated into thePNA of formula (I) at any suitable position in the PNA. Preferably, eachof R^(i) is a chemical moiety to which is attached a fluorescent group.It is more preferred that R^(i) is an amino acid that has beenderivatized with a fluorescent group. It is further more preferred thatR^(i) is a lysine with an ε-fluoresceinyl group.

[0049] Liposomal compositions of the invention comprise PNAs of theinvention which are incorporated into liposomes. The liposomalcompositions exhibit enhanced cellular uptake and distribution.Liposomes are a colloidal dispersion system, and constitute a stabledelivery system which protects the incorporated PNA from the environmentwhile being transported to target areas. Liposomes represent a stabledelivery vehicle to enhance the in vitro and in vivo stability of thePNAs of the invention. The liposomal compositions of the presentinvention, comprising PNAs of the invention incorporated into liposomes,can be formulated as pharmaceutical compositions according to standardtechniques known by the art-skilled using suitable and acceptablecarriers and adjuvants.

[0050] Liposomes that may be used include small unilamellar vesicles(SUVs), large unilamellar vesicles (LUVs) and multilamellar vesicles(MLVs). It has been shown that LUVs, which range in size from 0.2-0.4μm, can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules (e.g., RNA, DNA and intact virions canbe encapsulated within the aqueous interior and delivered to brain cellsin a biologically active form: Fraley et al., Trends Biochem. Sci.,1981, 6, 77). The composition of the liposome is usually a combinationof lipids, particularly phospholipids, in particular, high phasetransition temperature phospholipids, usually in combination with one ormore steroids, particularly cholesterol. Examples of lipids useful inliposome production include phosphatidyl compounds, such asphosphatidylglycerol, phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides.Particularly useful are diacyl phosphatidylglycerols, where the lipidmoiety contains from 14-18 carbon atoms, particularly from 16-18 carbonatoms, and is saturated (lacking double bonds within the 14-18 carbonatom chain). Illustrative phospholipids include phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0051] The targeting of liposomes can be either passive or active.Passive targeting utilizes the natural tendency of liposomes todistribute to cells of the reticuloendothelial system in organs thatcontain sinusoidal capillaries. Active targeting, by contrast, involvesmodification of the liposome by coupling thereto a specific ligand suchas a viral protein coat (Morishita et al., Proc. Natl. Acad. Sci.U.S.A., 1993, 90, 8474), monoclonal antibody (or a suitable bindingportion thereof), sugar, glycolipid or protein (or a suitableoligopeptide fragment thereof), or by changing the composition and/orsize of the liposome in order to achieve distribution to organs and celltypes other than the naturally occurring sites of localization. Thesurface of the targeted colloidal dispersion system can be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in close association with thelipid bilayer. Various linking groups can be used for joining the lipidchains to the targeting ligand. The targeting ligand, which binds aspecific cell surface molecule found predominantly on cells to whichdelivery of the oligonucleotides of the invention is desired, may be,for example, (1) a hormone, growth factor or a suitable oligopeptidefragment thereof which is bound by a specific cellular receptorpredominantly expressed by cells to which delivery is desired; or (2) apolyclonal or monoclonal antibody, or a suitable fragment thereof (e.g.,Fab; F(ab′)₂) which specifically binds an antigenic epitope foundpredominantly on targeted cells. Two or more bioactive agents (e.g., aPNA and a conventional drug, or two PNAs) can be combined within, anddelivered by, a single liposome. It is also possible to add agents tocolloidal dispersion systems which enhance the intercellular stabilityand/or targeting of the contents thereof.

[0052] The PNAs of the present invention may be used for gene modulation(e.g., gene targeted drugs), diagnostics, biotechnology and otherresearch purposes. The PNAs may also be used to target RNA andsingle-stranded DNA (ssDNA) to produce both antisense-type generegulating moieties and as hybridization probes, e.g., for theidentification and purification of nucleic acids. Furthermore, the PNAsmay be modified in such a way that they form triple helices with doublestranded DNA (dsDNA). Compounds that bind sequence-specifically to dsDNAhave applications as gene targeted drugs. These compounds are extremelyuseful drugs for treating various diseases, including cancer, acquiredimmune deficiency syndrome (AIDS) and other virus infections and geneticdisorders. Furthermore, these compounds may be used in research,diagnostics and for detection and isolation of specific nucleic acids.

[0053] Gene-targeted drugs are designed with a nucleobase sequence(preferably containing 10-20 units) complementary to the regulatoryregion (the promoter) of the target gene. Therefore, uponadministration, the gene-targeted drugs bind to the promoter and preventRNA polymerase from accessing the promoter. Consequently, no mRNA, andthus no gene product (protein), is produced. If the target is within avital gene for a virus, no viable virus particles will be produced.Alternatively, the target region could be downstream from the promoter,causing the RNA polymerase to terminate at this position, thus forming atruncated mRNA/protein which is nonfunctional.

[0054] Synthesis of PNA Oligomers

[0055] The principle of anchoring molecules during a reaction onto asolid matrix is known as Solid Phase Synthesis or Merrifield Synthesis.Merrifield, J. Am. Chem. Soc., 1963, 85, 2149; and Science, 1986, 232,341. Established methods for the stepwise or fragment-wise solid phaseassembly of amino acids into peptides normally employ a beaded matrix ofcross-linked styrene-divinylbenzene copolymer. The cross-linkedcopolymer is formed by the pearl polymerization of styrene monomer towhich is added a mixture of divinylbenzenes. Usually, 1-2% cross-linkingis employed. Such a matrix may be used in solid phase PNA synthesis ofthe present invention.

[0056] More than fifty methods for initial functionalization of thesolid phase have been described in connection with traditional solidphase peptide synthesis. Barany and Merrifield in “The Peptides,” Vol.2, Academic Press, New York, 1979, pp. 1; and Stewart and Young in“Solid Phase Peptide Synthesis,” 2nd ed., Pierce Chemical Company,Illinois, 1984. Regardless of its nature, the purpose of introducing afunctionality on the solid phase is to form an anchoring linkage betweenthe copolymer solid support and the C-terminus of the first amino acidto be coupled to the solid support. As will be recognized, anchoringlinkages may also be formed between the solid support and the amino acidN-terminus. The “concentration” of a functional group present in thesolid phase is generally expressed in millimoles per gram (mmol/g). Allof these established methods are, in principle, useful within thecontext of the present invention.

[0057] A preferred method for PNA synthesis employs aminomethyl as theinitial functionality. Aminomethyl is particularly advantageous as a“spacer” or “handle” group because it forms amide bonds with acarboxylic acid group in nearly quantitative amounts. A vast number ofrelevant spacer- or handle-forming bifunctional reagents have beendescribed. Barany et al., Int. J. Peptide Protein Res., 1987, 30, 705.Certain functionalities (e.g., benzhydrylamino, 4-methyl-benzhydrylaminoand 4-methoxybenzhydrylamino) which may be incorporated for the purposeof cleavage of a synthesized PNA chain from the solid support such thatthe C-terminal of the PNA chain is released as an amide, require nointroduction of a spacer group. Any such functionality mayadvantageously be employed in the context of the present invention.

[0058] Exemplary N-protecting groups are tert-butyloxycarbonyl (BLOC)(Carpino, J. Am. Chem. Soc., 1957, 79, 4427; McKay, et al., J. Am. Chem.Soc., 1957, 79, 4686; and Anderson et al., J. Am. Chem. Soc,, 1957, 79,6180), 9-fluorenylmethyloxycarbonyl (FMOC) (Carpino et al., J. Am. Chem.Soc., 1970, 92, 5748 and J. Org. Chem., 1972, 37, 3404), Adoc (Hass etal., J. Am. Chem. Soc., 1966, 88, 1988), Bpoc (Sieber Helv. Chem. Acta.,1968, 51, 614), Mcb (Brady et al., J. Org. Chem., 1977, 42, 143), Bic(Kemp et al., Tetrahedron, 1975, 4624), o-nitrophenylsulfenyl (Nps)(Zervas et al., J. Am. Chem. Soc., 1963, 85, 3660) and dithiasuccinoyl(Dts) (Barany et al., J. Am. Chem. Soc., 1977, 99, 7363), as well asother groups which are known to those skilled in the art. Theseamino-protecting groups, particularly those based on the widely-usedurethane functionality, prohibit racemization (mediated bytautomerization of the readily formed oxazolinone (azlactone)intermediates (Goodman et al., J. Am. Chem. Soc., 1964, 86, 2918))during the coupling of most α-amino acids. In addition to suchamino-protecting groups, nonurethane-type of amino-protecting groups arealso applicable when assembling PNA molecules.

[0059] The choice of side chain protecting groups, in general, dependson the choice of the amino-protecting group, because the side chainprotecting group must withstand the conditions of the repeated aminodeprotection cycles. This is true whether the overall strategy forchemically assembling PNA molecules relies on, for example, differentacid stability of amino and side chain protecting groups (such as is thecase for the above-mentioned “BOC-benzyl” approach) or employs anorthogonal, that is, chemoselective, protection scheme (such as is thecase for the above-mentioned “FMOC-t-Bu” approach).

[0060] Following coupling of the first amino acid, the next stage ofsolid phase synthesis is the systematic elaboration of the desired PNAchain. This elaboration involves repeated deprotection/coupling cycles.A temporary protecting group, such as BOC or FMOC, on the last coupledamino acid is quantitatively removed by a suitable treatment, forexample, by acidolysis, such as with trifluoroacetic acid in the case ofBOC, or by base treatment, such as with piperidine in the case of FMOC,so as to liberate the N-terminal amine function.

[0061] The next desired N-protected amino acid is then coupled to theN-terminal of the last coupled amino acid. This coupling of theC-terminal of an amino acid with the N-terminal of the last coupledamino acid can be achieved in several ways. For example, it can beachieved by providing the incoming amino acid in a form with thecarboxyl group activated by any of several methods, including theinitial formation of an active ester derivative such as a phthalimidoester (Nefkens et al., J. Am. Chem. Soc., 1961, 83, 1263), apentafluorophenyl ester (Kovacs et al., J. Am. Chem. Soc., 1963, 85,183), an imidazole ester (Li et al., J. Am. Chem. Soc., 1970, 92, 7608),and a 3-hydroxy-4-oxo-3,4dihydroquinazoline (Dhbt-OH) ester (Konig etal., Chem. Ber., 1973, 103, 2024 and 2034), or the initial formation ofan anhydride such as a symmetrical anhydride (Wieland et al., Angew.Chem., Int. Ed. Engl., 1971, 10, 336). Alternatively, the carboxyl groupof the incoming amino acid can be reacted directly with the N-terminalof the last coupled amino acid with the assistance of a condensationreagent such as, for example, dicyclohexylcarbodiimide (Sheehan et al.,J. Am. Chem. Soc., 1955, 77, 1067) or derivatives thereof.Benzotriazolyl N-oxy-trisdimethylaminophosphonium hexafluorophosphate(BOP), “Castro's reagent” (see Rivaille et al., Tetrahedron, 1980, 36,3413), is recommended when assembling PNA molecules containing secondaryamino groups.

[0062] Following the assembly of the desired PNA chain, includingprotecting groups, the next step will normally be deprotection of theamino acid moieties of the PNA chain and cleavage of the synthesized PNAfrom the solid support. These processes can take place substantiallysimultaneously, thereby providing the free PNA molecule in the desiredform.

[0063] In the above-mentioned “BOC-benzyl” protection scheme, the finaldeprotection of side chains and release of the PNA molecule from thesolid support is most often carried out by the use of strong acids suchas anhydrous HF (Sakakibara et al., Bull. Chem. Soc. Jpn., 1965, 38,4921), boron tris (trifluoroacetate) (Pless et al., Helv. Chim. Acta,1973, 46, 1609) and sulfonic acids, such as trifluoromethanesulfonicacid and methanesulfonic acid (Yajima et al., J. Chem. Soc., Chem.Comm., 1974, 107). A strong acid (e.g., anhydrous HF) deprotectionmethod may produce very reactive carbocations that may lead toalkylation and acylation of sensitive residues in the PNA chain. Suchside reactions are only partly avoided by the presence of scavengerssuch as anisole, phenol, dimethyl sulfide, and mercaptoethanol. Thus,the sulfide-assisted acidolytic S_(N)2 deprotection method (Tam et al.,J. Am. Chem. Soc., 1983, 105, 6442 and J. Am. Chem. Soc., 1986, 108,5242), the so-called “low” method, which removes the precursors ofharmful carbocations to form inert sulfonium salts, is frequentlyemployed in peptide and PNA synthesis. Other methods for deprotectionand/or final cleavage of the PNA-solid support bond may includebase-catalyzed alcoholysis (Barton et al., J. Am. Chem. Soc., 1973, 95,4501), ammonolysis, hydrazinolysis (Bodanszky et al., Chem. Ind., 19641423), hydrogenolysis (Jones, Tetrahedron Lett. 1977 2853 and Schlatteret al., Tetrahedron Lett. 1977 2861)) and photolysis (Rich and Gurwara,J. Am. Chem. Soc., 1975 97, 1575)).

[0064] Finally, in contrast with the chemical synthesis of conventionalpeptides, stepwise chain building of achiral PNAs such as those based onaminoethylglycyl backbone units can start either from the N-terminus orthe C-terminus. Those skilled in the art will recognize that synthesiscommencing at the C-terminus typically employ protected amine groups andfree or activated acid groups, and syntheses commencing at theN-terminus typically employ protected acid groups and free or activatedamine groups.

[0065] Likely therapeutic and prophylactic targets include herpessimplex virus (HSV), human papillomavirus (HPV), human immunodeficiencyvirus (HIV), candida albicans, influenza virus, cytomegalovirus (CMV),intercellular adhesion molecules (ICAM), 5-lipoxygenase (5-LO),phospholipase A₂ (PLA₂), protein kinase C (PKC), and the ras oncogene.Potential treatment of such targeting include ocular, labial, genital,and systemic herpes simplex I and II infections; genital warts; cervicalcancer, common warts; Kaposi's sarcoma; AIDS; skin and systemic fungalinfections; flu; pneumonia; retinitis and pneumonitis inimmunosuppressed patients; mononucleosis; ocular, skin and systemicinflammation; cardiovascular disease; cancer; asthma; psoriasis;cardiovascular collapse; cardiac infarction; gastrointestinal disease;kidney disease; rheumatoid arthritis; osteoarthritis; acutepancreatitis; septic shock; and Crohn's disease.

[0066] In general, for therapeutic or prophylactic treatment, a patientsuspected of requiring such therapy is administered a PNA or liposomalcomposition of the present invention, commonly in a pharmaceuticallyacceptable carrier, in amounts and for periods of time which will varydepending upon the nature of the particular disease, its severity andthe patient's overall condition. The PNAs and liposomal compositions ofthe invention can be formulated in a pharmaceutical composition, whichmay include carriers, thickeners, diluents, buffers, preservatives,surface active agents and the like. Pharmaceutical compositions may alsoinclude one or more active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics and the like, in addition to thepeptide nucleic acids.

[0067] The pharmaceutical composition may be administered in a number ofways depending upon whether local or systemic treatment is desired, andupon the area to be treated. Administration may be topical (includingophthalmic, vaginal, rectal, intranasal, transdermal), oral orparenteral, for example, by intravenous drip, subcutaneous,intraperitoneal or intramuscular injection or intrathecal orintraventricular administration.

[0068] Formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, nucleic acidcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable in certain circumstances. Coated condoms, glovesand the like may also be useful. Topical administration also includesdelivery of the PNAs and liposomal compositions of the invention intothe epidermis of an animal by electroporation. Zewart et al. WO96/39531, published Dec. 12, 1996.

[0069] Compositions for oral administration include powders or granules,suspensions or solutions in aqueous or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

[0070] Intravitreal injection, for direct delivery of the PNAs andliposomal compositions of the invention to the vitreous humor of the eyeof an animal is described in U.S. Pat. No. 5,595,978, issued Jan. 21,1997, the contents of which are herein incorporated by reference.

[0071] Intraluminal administration, for direct delivery of PNAs andliposomal compositions of the invention to an isolated portion of atubular organ or tissue (e.g., artery, vein, ureter or urethra) may bedesired for the treatment of patients with diseases or conditionsafflicting the lumen of such organs or tissues. To effect this mode ofadministration, a catheter or cannula is surgically introduced byappropriate means. After isolation of the portion of the tubular organor tissue for which treatment is sought, the PNA or liposomalcomposition of the invention is infused through the catheter or cannula.The infusion catheter or cannula is then removed, and flow within thetubular organ or tissue is restored by removal of the ligatures whicheffected the isolation of a segment thereof. Morishita et al., Proc.Natl. Acad. Sci., U.S.A., 1993, 90, 8474.

[0072] Intraventricular administration, for direct delivery of PNAs orliposomal compositions of the invention to the brain of a patient, maybe desired for the treatment of patients with diseases or conditionsafflicting the brain. To effect this mode of administration, a siliconcatheter is surgically introduced into a ventricle of the brain, and isconnected to a subcutaneous infusion pump (Medtronic, Inc., Minneapolis,Minn.) that has been surgically implanted in the abdominal region. Zimmet al., Cancer Research, 1984, 44, 1698; and Shaw, Cancer, 1993, 72(11Suppl.), 3416. The pump is used to inject the PNA or liposomalcomposition, and allows precise dosage adjustments and variation indosage schedules with the aid of an external programming device. Thereservoir capacity of the pump is 18-20 mL, and infusion rates may rangefrom 0.1 mL/hour to 1 mL/hour. Depending on the frequency ofadministration, ranging from daily to monthly, and the dose to beadministered, ranging from 0.01 μg to 100 g per kg of body weight, thepump reservoir may be refilled at 3-10 week intervals. Refilling of thepump is accomplished by percutaneous puncture of the self-sealing septumof the pump. Compositions for intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

[0073] Intrathecal administration, for the direct delivery of PNAs orliposomal compositions of the invention into the spinal column of apatient, may be desired for the treatment of patients with diseases ofthe central nervous system. To effect this route of administration, asilicon catheter is surgically implanted into the L3-4 lumbar spinalinterspace of the patient, and is connected to a subcutaneous infusionpump which has been surgically implanted in the upper abdominal region.Luer and Hatton, The Annals of Pharmacotherapy, 1993, 27, 912; Ettingeret al., Cancer, 1978, 41, 1270; and Yaida et al., Regul. Pept., 1995,59, 193. The pump is used to inject the PNA or liposomal composition,and allows precise dosage adjustments and variations in dose scheduleswith the aid of an external programming device. The reservoir capacityof the pump is 18-20 mL, and infusion rates may vary from 0.1 mL/hour to1 mL/hour. Depending on the frequency of administration, ranging fromdaily to monthly, and dosage to be administered, ranging from 0.01 μg to100 g per kg of body weight, the pump reservoir may be refilled at 3-10week intervals. Refilling of the pump is accomplished by a singlepercutaneous puncture to the self-sealing septum of the pump.Compositions for intrathecal administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

[0074] To effect delivery to areas other than the brain or spinal columnvia this method, the silicon catheter may be configured to connect thesubcutaneous infusion pump to, e.g., the hepatic artery, for delivery tothe liver. Kemeny et al., Cancer, 1993, 71, 1964. Infusion pumps mayalso be used to effect systemic delivery. Ewel et al., Cancer Research,1992, 52, 3005; and Rubenstein et al., J. Surg. Oncol., 1996, 62, 194.

[0075] Compositions for parenteral, intrathecal or intraventricularadministration, or liposomal systems, may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual PNAs, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years.

[0076] Synthesis of Monomer Subunits.

[0077] The monomer subunits preferably are synthesized by a generalscheme that commences with the preparation of either the methyl or ethylester of (BOC-aminoethyl)glycine, via a protection/deprotectionprocedure, as described in Examples 1 and 2. The synthesis of thyminemonomer is described in Examples 4 and 5, and the synthesis of protectedcytosine monomer is described in Example 6.

[0078] The synthesis of a protected adenine monomer involves alkylationof adenine with ethyl bromoacetate (Example 7) and verification of theposition of substitution (i.e. position 9) by X-ray crystallography. TheN⁶-amino group is then protected with the benzyloxycarbonyl group by theuse of the reagent N-ethyl-benzyloxycarbonylimidazole tetrafluoroborate(Example 8). Simple hydrolysis of the product ester (Example 9) gaveN⁶-benzyloxycarbonyl-9-carboxymethyl adenine (Examples 10 and 11), whichwas used in the standard PNA oligomer synthesis.

[0079] For the synthesis of the protected G-monomer, the startingmaterial, 2-amino-6-chloro-purine, was alkylated with bromoacetic acid(Example 12), and the 6-chloro group was then substituted with abenzyloxy group (Example 13). The resulting acid was coupled to the(BOC-aminoethyl)glycine methyl ester (from Example 2) with agent PyBrop™being used as a coupling agent, and the resulting ester was hydrolyzed(Example 14) to afford the protected G monomer. The O⁶-benzyl group wasremoved in the final HF-cleavage step following synthesis of thePNA-oligomer.

[0080] Additional objects, advantages, and novel features of the presentinvention will become apparent to those skilled in the art uponexamination of the following examples thereof. The following examplesillustrate the invention and are not intended to limit the same. Thoseskilled in the art will recognize, or be able to ascertain throughroutine experimentation, numerous equivalents to the specificsubstances, compositions, and procedures described herein. Suchequivalents are considered to be within the scope of the presentinvention.

[0081] General Remarks.

[0082] The following abbreviations are used in the experimentalexamples: DMF, N,N-dimethylformamide; Tyr, tyrosine; Lys, lysine; DCC,N,N-dicyclohexyl-carbodiimide; DCU, N,N-dicyclohexylurea; THF,tetrahydrofuran; aeg, N-acetyl-N-(2′-aminoethyl)glycine; aek,N-acetyl-N-(2′-aminoethyl)lysine; Pfp, pentafluorophenyl; BOC,tert-butoxycarbonyl; Z, benzyloxycarbonyl; NMR, nuclear magneticresonance; s, singlet; d, doublet; dd, doublet of doublets; t; triplet;q, quartet; m, multiplet; b, broad; δ, chemical shift; ppm, parts permillion (chemical shift).

[0083] NMR spectra were recorded on JEOL FX 90Q spectrometer or a Bruker250 MHz with tetramethylsilane as an internal standard. Massspectrometry was performed on a MassLab VG 12-250 quadropole instrumentfitted with a VG FAB source and probe. Melting points were recorded on aBuchi melting point apparatus and are uncorrected. N,N-Dimethylformamidewas dried over 4 Å molecular sieves, distilled and stored over 4 Åmolecular sieves. Pyridine (HPLC quality) was dried and stored over 4 Åmolecular sieves. Other solvents used were either the highest qualityobtainable or were distilled prior to use. Dioxane was passed throughbasic alumina prior to use. BOC-anhydride, 4-nitrophenol, methylbromoacetate, benzyloxycarbonyl chloride, pentafluorophenol were allobtained from Aldrich Chemical Company. Thymine, cytosine, adenine wereall obtained from Sigma.

[0084] Thin layer chromatography (tic) was performed using the followingsolvent systems: (1) chloroform:triethyl amine:methanol, 7:1:2; (2)methylene chloride:methanol, 9:1; (3) chloroform:methanol:acetic acid85:10:5. Spots were visualized by UV (254 nm) and/or spraying with aninhydrin solution (3 g ninhydrin in 1000 mL of 1-butanol and 30 mL ofacetic acid), after heating at 120° C. for 5 minutes and, afterspraying, heating again.

[0085] The carboxyl terminal (C terminus) end of PNA oligomers can besubstituted with a variety of functional groups. One way this isperformed is through the use of different resins. The amino terminal (Nterminus) end of PNA oligomers can also be capped with a carboxylicacid-based capping reagent for the final PNA monomer in the finalcoupling step, or substituted with a variety of conjugate groups.Representative examples of the types of C and N terminal groups areshown below. Resin Employed aeg-PNA/aeg-PNA Derivative Prepared (CappingReagent = Acetyl) Merrifield CH₃CONH-(PNA)-COOH H₂N-(PNA)-COOH LysSubstituted Merifield H₂N-(PNA)-Lys-COOH Merrifield H₂N-(PNA)-CONH₂ LysSubstituted MBHA H₂N-(PNA)-Lys-CONH₂ Lys Substituted MerrifieldCH₃CONH-(PNA)-Lys-COOH H₂N-(PNA)-COOH Lys Substituted MerrifieldH₂N-(PNA)-Lys-COOH Merrifield H₂N-(PNA)-CONH₂ MBHA H₂N-(PNA)-CONH₂ LysSubstituted MBHA H₂N-(PNA)-Lys-CONH₂ MBHA CH₃CONH-(PNA)-CONH₂H₂N-(PNA)-CONH₂ Lys Substituted MBHA CH₃CONH-(PNA)-Lys-CONH₂ (CappingReagent = N-Boc glycine) Merrifield BocGly-(PNA)-COOH Lys SubstitutedMerrifield BocGly-(PNA)-Lys-COOH MBHA BocGly-(PNA)-CONH₂ Lys SubstitutedMBHA BocGly-(PNA)-Lys-CONH₂ (Capping Reagent = 1. Glycine; 2. CholicAcid (Chol)) Merrifield Chol-Gly-(PNA)-COOH Lys Substituted MerrifieldChol-Gly-(PNA)-Lys-COOH MBHA Chol-Gly-(PNA)-CONH₂ Lys Substituted MBHAChol-Gly-(PNA)-Lys-CONH₂

[0086] Further examples are found in U.S. application Ser. No.08/275,951, filed Jul. 15, 1994, and incorporated herein by reference.

EXAMPLE 1 Synthesis of N-Benzyloxycarbonyl-N-′(BOC-Aminoethyl)Glycine.

[0087] Aminoethyl glycine (52.86 g, 0.447 mol) was dissolved in water(900 mL) and dioxane (900 mL) was added. The pH was adjusted to 11.2with 2N NaOH. While the pH was kept at 11.2, tert-butyl-p-nitrophenylcarbonate (128.4 g, 0.537 mol) was dissolved in dioxane (720 mL) andadded dropwise over the course of 2 hours. The pH was kept at 11.2 forat least three more hours and then allowed to stand overnight, withstirring. The yellow solution was cooled to 0° C. and the pH wasadjusted to 3.5 with 2 N HCl. The mixture was washed with chloroform(4×100 mL), and the pH of the aqueous phase was readjusted to 9.5 with 2N NaOH at 0° C. Benzyloxycarbonyl chloride (73.5 mL, 0.515 mol) wasadded over half an hour, while the pH was kept at 9.5 with 2 N NaOH. ThepH was adjusted frequently over the next 4 hours, and the solution wasallowed to stand overnight, with stirring. On the following day thesolution was washed with ether (3×600 mL) and the pH of the solution wasafterwards adjusted to 1.5 with 2 N HCl at 0° C. The title compound wasisolated by extraction with ethyl acetate (5×1000 mL). The ethyl acetatesolution was dried over magnesium sulfate and evaporated to dryness, invacuo. This afforded 138 g of the product, which was dissolved in ether(300 mL) and precipitated by the addition of petroleum ether (1800 mL).Yield 124.7 g (79%). M.p. 64.5-85 ° C. Anal. for C₁₇H₂₄N₂O₆ found(calc.)C: 58.40(57.94); H: 7.02(6.86); N: 7.94(7.95). ¹H—NMR (250 MHz, CDCl₃)7.33 & 7.32 (5H, Ph); 5.15 & 5.12 (2H, PhCH ₂); 4.03 & 4.01 (2H, NCH₂CO₂H); 3.46 (b, 2H, BOC—NHCH₂CH ₂); 3.28 (b, 2H, BOC—NHCH ₂CH₂); 1.43 &1.40 (9H, t-Bu). HPLC (260 nm) 20.71 min. (80.2%) and 21.57 min.(19.8%). The UV-spectra (200 nm-300 nm) are identical, indicating thatthe minor peak consists of Bis-Z-AEG.

EXAMPLE 2 Synthesis of N′-BOC-Aminoethylglycine Esters

[0088] (a) Ethyl ester: N-Benzyloxycarbonyl-N′-(BOC-aminoethyl)glycine(60 g, 0.170 mol) and N,N-dimethyl-4-amninopyridine (6 g) were dissolvedin absolute ethanol (500 mL), and cooled to 0° C. before the addition ofDCC (42.2 g, 0.204 mol). The ice bath was removed after 5 minutes andstirring was continued for 2 more hours. The precipitated DCU (32.5 g,dried) was removed by filtration and washed with ether (3×100 mL). Thecombined filtrate was washed successively with diluted potassiumhydrogen sulfate (2×400 mL), diluted sodium hydrogencarbonate (2×400 mL)and saturated sodium chloride (1×400 mL). The organic phase wasfiltered, then dried over magnesium sulfate, and evaporated to dryness,in vacuo, which yielded 66.1 g of an oily substance which contained someDCU.

[0089] The oil was dissolved in absolute ethanol (600 mL) and was added10% palladium on carbon (6.6 g) was added. The solution was hydrogenatedat atmospheric pressure. After 4 hours, 3.3 L was consumed out of thetheoretical 4.2 L. The reaction mixture was filtered through celite andevaporated to dryness, in vacuo, affording 39.5 g (94%) of an oilysubstance. A 13 g portion of the oily substance was purified by silicagel (SiO₂, 600 g) chromatography. After elution with 300 mL of 20%petroleum ether in methylene chloride, the title compound was elutedwith 1700 mL of 5% methanol in methylene chloride. The solvent wasremoved from the fractions, in vacuo, to yield 8.49 g product ofsatisfactory purity. Alternatively 10 g of the crude material waspurified by Kugelrohr distillation. ¹H—NMR (250 MHz, CD₃OD); 4.77 (b. s,NH); 4.18 (q, 2H, MeCH ₂—); 3.38 (s, 2H, NCH ₂CO₂Et); 3.16 (t, 2H,BOC—NHCH ₂CH₂); 2.68 (t, 2H, BOC—NHCH₂CH ₂); 1.43 (s, 9H, t-Bu) and 1.26(t, 3H, CH₃)¹³C—NMR 171.4 (COEt); 156.6 (CO); 78.3 ((CH₃)₃ C); 59.9(CH₂); 49.0 (CH₂); 48.1 (CH₂); 39.0 (CH₂); 26.9 (CH₂) and 12.6 (CH₃).

[0090] (b) Methyl ester: The above procedure for the ethyl ester wasused, with methanol being substituted for ethanol. The final product waspurified by column chromatography.

EXAMPLE 3 Alternate Large-Scale Synthesis of (N′-BOC-Aminoethyl)GlycineEthyl Ester

[0091] (a) Preparation of BOC-aminoacetaldehyde.

[0092] 3-Amino-1,2-propanediol (80 g, 0.88 mol) was dissolved in water(1500 mL) and the solution was cooled to 4° C., after whichBOC-anhydride (230 g, 1.05 mol) was added in one portion. The solutionwas gently heated to room temperature in a water bath. The pH wasmaintained at 10.5 by the dropwise addition of sodium hydroxide. Overthe course of the reaction, a total of 70.2 g of NaOH, dissolved in 480mL of water, was added. After stirring overnight, ethyl acetate (1000mL) was added, the mixture cooled to 0° C. and the pH adjusted to 2.5 bythe addition of 4 M hydrochloric acid. The ethyl acetate layer wasremoved and the acidic aqueous solution was extracted with more ethylacetate (8×500 mL). The combined ethyl acetate solution was reduced to avolume of 1500 mL using a rotary evaporator. The resulting solution waswashed with half saturated potassium hydrogen sulphate (1500 mL) andthen with saturated sodium chloride. It then was dried over magnesiumsulphate and evaporated to dryness, in vacuo. Yield: 145.3 g (86%).

[0093] 3-BOC-amino-1,2-propanediol (144.7 g, 0.757 mol) was suspended inwater (750 mL) and potassium periodate (191.5 g, 0.833 mol) was added.The mixture was stirred under nitrogen for 2.5 h and the precipitatedpotassium iodate was removed by filtration and washed once with water(100 mL). The aqueous phase was extracted with chloroform (6×400 mL).The chloroform extracts were dried and evaporated to dryness, in vacuo.Yield: 102 g (93%) of an oil. BOC-aminoacetaldehyde was purified bykugelrohr distillation at 84° C. and 0.3 mm Hg, in two portions. Yield:79 g (77%) as a colorless oil.

[0094] (b) Preparation of (N′-BOC-aminoethyl)glycine Methyl Ester.

[0095] Palladium on carbon (10%, 2.00 g) was added to a solution ofBOC-aminoacetaldehyde (10 g, 68.9 mmol) in methanol (150 mL) at 0° C.Sodium acetate ( 11.3 g, 138 mmol) in methanol (150 mL), and glycinemethyl ester hydrochloride (8.65 g; 68.9 mmol) in methanol (75 mL) wereadded. The mixture was hydrogenated at atmospheric pressure for 2.5 h,then filtered through celite and evaporated to dryness, in vacuo. Thematerial was redissolved in water (150 mL) and the pH adjusted to 8 with0.5 N NaOH. The aqueous solution was extracted with methylene chloride(5×150 mL). The combined extracts were dried over sodium sulphate andevaporated to dryness, in vacuo. This resulted in 14.1 g (88%) yield of(N′-BOC-aminoethyl)glycine methyl ester. The crude material was purifiedby kugelrohr destination at 120° C. and 0.5 mm Hg to give 11.3 g (70%)of a colorless oil. The product had a purity that was higher than thematerial produced in example 26 according to tlc analysis (10% methanolin methylene chloride).

[0096] Alternatively, sodium cyanoborohydride can be used as reducingagent instead of hydrogen (with Pd(C) as catalyst), although the yield(42%) was lower.

[0097] (c) Preparation of (N′-BOC-aminoethyl)glycine Ethyl Ester.

[0098] The title compound was prepared by the above procedure withglycine ethyl ester hydrochloride substituted for glycine methyl esterhydrochloride. Also, the solvent used was ethanol. The yield was 78%.

EXAMPLE 4 Synthesis of 1(BOC-aeg)Thymine Ethyl Ester

[0099] N′-BOC-aminoethylglycine ethyl ester (13.5 g, 54.8 mmol), DhbtOH(9.84 g, 60.3 mmol) and 1-carboxymethyl thymine (11.1 g, 60.3 mmol) weredissolved in DMF (210 mL). Methylene chloride (210 mL) was added. Thesolution was cooled to 0° C. in an ethanol/ice bath and DCC (13.6 g,65.8 mmol) was added. The ice bath was removed after 1 hour and stirringwas continued for another 2 hours at ambient temperature. Theprecipitated DCU was removed by filtration and washed twice withmethylene chloride (2×75 mL). To the combined filtrate was added moremethylene chloride (650 mL). The solution was washed successively withdiluted sodium hydrogen carbonate (3×500 mL), diluted potassium hydrogensulfate (2×500 mL), and saturated sodium chloride (1×500 mL). Some ofthe precipitate was removed from the organic phase by filtration. Theorganic phase was dried over magnesium sulfate and evaporated todryness, in vacuo. The oily residue was dissolved in methylene chloride(150 mL), filtered, and the title compound was precipitated by theaddition of petroleum ether (350 mL) at 0° C. The methylene chloridepetroleum ether procedure was repeated once. This afforded 16 g (71%) ofa material which was more than 99% pure by HPLC.

EXAMPLE 5 Synthesis of (BOC-aeg)Thymine.

[0100] The material from Example 4 was suspended in THF (194 mL, gives a0.2 M solution), and 1 M aqueous lithium hydroxide (116 mL) was added.The mixture was stirred for 45 minutes at ambient temperature and thenfiltered to remove residual DCU. Water (40 mL) was added to the solutionwhich was then washed with methylene chloride (300 mL). Additional water(30 mL) was added, and the alkaline solution was washed once more withmethylene chloride (150 mL). The aqueous solution was cooled to 0° C.and the pH was adjusted to 2 by the dropwise addition of 1 N HCl(approx. 110 mL). The title compound was extracted with ethyl acetate(9×200 mL), the combined extracts were dried over magnesium sulfate andwere evaporated to dryness, in vacuo. The residue was evaporated oncefrom methanol, which after drying overnight afforded a colorless glassysolid. Yield: 9.57 g (64%). HPLC>98% R_(T)=14.8 minutes. Anal. forC₁₆H₂₄N₄O₇ _(^(o)) 0.25 H₂O Found (calc.) C: 49.29(49.42); H:6.52(6.35); N: 14.11(14.41). Due to the limited rotation around thesecondary amide, several of the signals were doubled in the ratio 2:1(indicated in the list by mj. for major and mi. for minor). ¹H—NMR (250MHz, DMSO-d₆) δ: 12.75 (bs, 1H, CO₂H); 11.28 (s, 1H, mj, imide NH);11.26 (s, 1H, mi, imide NH); 7.30 (s, 1H, mj, T H-6); 7.26 (s, 1H, mi, TH-6); 6.92 (bt, 1H, mj, BOC—NH); 6.73 (bt, 1H, mi, BOC—NH); 4.64 (s, 2H,mj, CH ₂CON); 4.46 (s, 2H, mj, CH ₂CON); 4.19 (s, 2H, mi, CH ₂CO₂H);3.97 (s, 2H, mj, CH ₂CO₂H); 3.63-3.01 (unresolved m, includes water, CH₂CH ₂); 1.75 (s, 3H, CH ₃) and 1.38 (s, 9H, t-Bu).

EXAMPLE 6 Synthesis of N⁴-Benzyloxycarbonyl-1-(BOC-aeg)Cytosine.

[0101] N′-BOC-aminoethyl glycine ethyl ester (5 g, 20.3 mmol), DhbtOH(3.64 g, 22.3 mmol) and N⁴-benzyloxycarbonyl-1-carboxymethyl cytosine(6.77 g, 22.3 mmol) were suspended in DMF (100 mL). Methylene chloride(100 mL) was added. The solution was cooled to 0° C. and DCC (5.03 g,24.4 mmol) was added. The ice bath was removed after 2 h and stirringwas continued for another hour at ambient temperature. The reactionmixture then was evaporated to dryness, in vacuo. The residue wassuspended in ether (100 mL) and stirred vigorously for 30 minutes. Thesolid material was isolated by filtration and the ether wash procedurewas repeated twice. The material was then stirred vigorously for 15minutes with dilute sodium hydrogencarbonate (approx. 4% solution, 100mL), filtered and washed with water. This procedure was then repeatedonce, which after drying left 17 g of yellowish solid material. Thesolid was then refluxed with dioxane (200 mL) and filtered while hot.After cooling, water (200 mL) was added. The precipitated material wasisolated by filtration, washed with water, and dried. According to HPLC(observing at 260 nm) this material has a purity higher than 99%,besides the DCU. The ester was then suspended in THF (100 mL), cooled to0° C., and 1 N LiOH (61 mL) was added. After stirring for 15 minutes,the mixture was filtered and the filtrate was washed with methylenechloride (2×150 mL). The alkaline solution then was cooled to 0° C. andthe pH was adjusted to 2.0 with 1 N HCl. The title compound was isolatedby filtration and was washed once with water, leaving 11.3 g of a whitepowder after drying. The material was suspended in methylene chloride(300 mL) and petroleum ether (300 mL) was added. Filtration and washafforded 7.1 g (69%) after drying. HPLC showed a purity of 99%R_(T)=19.5 minutes, and a minor impurity at 12.6 minutes (approx. 1%)most likely the Z-deprotected monomer. Anal. for C₂₃H₂₉N₅O₈ found(calc.)C: 54.16(54.87); H: 5.76(5.81) and N: 13.65(13.91). ¹H—NMR (250 MHZ,DMSO-d₆). 10.78 (bs, 1H, CO₂ H) 7.88 (2 overlapping doublets, 1H, CytH-5); 7.41-7.32 (m, 5H, Ph); 7.01 (2 overlapping doublets, 1H, Cyt H-6);6.94 & 6.78 (unresolved triplets, 1H, BOC—NH); 5.19 (s, 2H, PhCH ₂);4.81 & 4.62 (s, 2H, CH ₂CON); 4.17 & 3.98 (s, 2H, CH ₂CO₂H); 3.42-3.03(m, includes water, CH ₂CH ₂) and 1.38 & 1.37 (s, 9H, ^(t)-Bu). ¹³C—NMR.150.88; 128.52; 128.18; 127.96; 93.90; 66.53; 49.58 and 28.22. IR:Frequency in cm⁻¹. 3423, 3035, 2978, 1736, 1658, 1563, 1501 and 1456.

EXAMPLE 7 Synthesis of 9-Carboxymethyladenine Ethyl Ester.

[0102] Adenine (10 g, 74 mmol) and potassium carbonate (10.29 g, 74mmol) were suspended in DMF and ethyl bromoacetate (8.24 mL, 74 mmol)was added. The suspension was stirred for 2.5 h under nitrogen at roomtemperature and then filtered. The solid residue was washed three timeswith DMF (10 mL). The combined filtrate was evaporated to dryness, invacuo. Water (200 mL) was added to the yellowish-orange solid materialand the pH adjusted to 6 with 4 N HCl. After stirring at 0° C. for 10minutes, the solid was filtered off, washed with water, andrecrystallized from 96% ethanol (150 mL). The title compound wasisolated by filtration and washed thoroughly with ether. Yield: 3.4 g(20%). M.p. 215.5-220° C. Anal. for C₉H₁₁N₅O₂ found(calc.): C:48.86(48.65); H: 5.01(4.91); N: 31.66(31.42). ¹H—NMR (250 MHZ; DMSO-d₆):7.25 (bs, 2H, NH₂), 5.06 (s, 2H, NCH₂),4.17 (q, 2H, J=7.11 Hz, OCH₂) and1.21 (t, 3H, J=7.13 Hz, NCH₂). ¹³C—NMR. 152.70, 141.30, 61.41, 43.97 and14.07. FAB-MS. 222 (MH+). IR: Frequency in cm⁻¹. 3855, 3274, 3246, 3117,2989, 2940, 2876, 2753, 2346, 2106, 1899, 1762, 1742, 1742, 1671, 1644,1606, 1582, 1522, 1477, 1445 and 1422. The position of alkylation wasverified by X-ray crystallography on crystals, which were obtained byrecrystallization from 96% ethanol.

[0103] Alternatively, 9-carboxymethyladenine ethyl ester can be preparedby the following procedure. To a suspension of adenine (50 g, 0.37 mol)in DMF (1100 mL) in 2 L three-necked flask equipped with a nitrogeninlet, a mechanical stir and a dropping funnel, was added 16.4 g (0.407mol) of hexane-washed sodium hydride-mineral oil dispersion. The mixturewas stirred vigorously for 2 hours, after which ethyl bromoacetate (75mL, 0.67 mol) was added dropwise over the course of 3 hours. The mixturewas stirred for one additional hour, after which tlc indicated completeconversion of adenine. The mixture was evaporated to dryness at 1 mm Hgand water (500 mL) was added to the oily residue which causedcrystallization of the title compound. The solid was recrystallized from96% ethanol (600 mL). Yield (after drying): 53.7 g (65.6%). HPLC (215nm) purity>99.5%.

EXAMPLE 8 Synthesis of N⁶⁻Benzyloxycarbonyl-9-Carboxymethyladenine EthylEster

[0104] 9-Carboxymethyladenine ethyl ester (3.4 g, 15.4 mmol) wasdissolved in dry DMF (50 mL) by gentle heating, cooled to 20° C., andadded to a solution of N-ethyl-benzyloxycarbonylimidazoletetrafluoroborate (62 mmol) in methylene chloride (50 mL) over a periodof 15 minutes in an ice bath. Some precipitation was observed. The icebath was removed and the solution was stirred overnight. The reactionmixture was treated with saturated sodium hydrogen carbonate (100 mL).After stirring for 10 minutes, the phases were separated and the organicphase was washed successively with one volume of water, dilute potassiumhydrogen sulfate (twice), and with saturated sodium chloride. Thesolution was dried over magnesium sulfate and evaporated to dryness, invacuo, which afforded 11 g of an oily material. The material wasdissolved in methylene chloride (25 mL), cooled to 0° C., andprecipitated with petroleum ether (50 mL). This procedure was repeatedonce to give 3.45 g (63%) of the title compound. M.p. 132-35° C.Analysis for C₁₇H₁₇N₅O₄ found (calc.): C: 56.95(57.46); H: 4.71(4.82);N: 19.35(19.71). ¹H—NMR (250 MHZ; CDCl₃): 8.77 (s, 1H, H-2 or H-8); 7.99(s, 1H, H-2 or H-8); 7.45-7.26 (m, 5H, Ph); 5.31 (s, 2H, N—CH ₂); 4.96(s, 2H, Ph-CH ₂); 4.27 (q, 2H, J=7.15 Hz, CH ₂CH₃) and 1.30(t,3H,J=7.15Hz, CH₂CH ₃). ¹³C—NMR: 153.09; 143.11; 128.66; 67.84; 62.51; 44.24 and14.09. FAB-MS: 356 (MH+) and 312 (MH+—CO₂). IR: frequency in cm⁻¹: 3423;3182; 3115; 3031; 2981; 1747; 1617; 15.87; 1552; 1511; 1492; 1465 and1413.

EXAMPLE 9 Synthesis of N⁶⁻Benzyloxycarbonyl-9-Carboxymethyladenine

[0105] N⁶-Benzyloxycarbonyl-9-carboxymethyladenine ethyl ester (3.2 g,9.01 mmol) was mixed with methanol (50 mL) cooled to 0° C. Sodiumhydroxide solution (2 N, 50 mL) was added, whereby the material quicklydissolved. After 30 minutes at 0° C., the alkaline solution was washedwith methylene chloride (2×50 mL). The pH of the aqueous solution wasadjusted to 1 with 4 N HCl at 0° C., whereby the title compoundprecipitated. The yield after filtration, washing with water, and dryingwas 3.08 g (104%). The product contained salt, and the elementalanalysis reflected that. Anal. for C₁₅H₁₃N₅O₄ found(calc.): C:46.32(55.05); H: 4.24(4.00); N: 18.10(21.40) and C/N: 2.57(2.56).¹H—NMR(250 MHZ; DMSO-d₆): 8.70 (s, 2H, H-2 and H-8); 7.50-7.35 (m, 5H,Ph); 5.27 (s, 2H, N—CH ₂); and 5.15 (s, 2H, Ph-CH ₂). ¹³C—NMR. 168.77,152.54 151.36, 148.75, 145.13, 128.51, 128.17, 127.98, 66.76 and44.67.IR (KBr) 3484; 3109; 3087; 2966; 2927; 2383; 1960; 1739; 1688;1655; 1594; 1560; 1530; 1499; 1475; 1455; 1429 and 1411. FAB-MS: 328(MH+) and 284 (MH+—CO₂). HPLC (215 nm, 260 nm) in system 1: 15.18 min.minor impurities all less than 2%.

EXAMPLE 10 Synthesis of N⁶-Benzyloxycarbonyl-1-(BOC-aeg)Adenine EthylEster

[0106] N′-BOC-aminoethylglycine ethyl ester (2 g, 8.12 mmol), DhbtOH(1.46 g, 8.93 mmol) and N⁶-benzyloxycarbonyl-9carboxymethyl adenine(2.92 g, 8.93 mmol) were dissolved in DMF (15 mL). Methylene chloride(15 mL) was then added. The solution was cooled to 0° C. in anethanol/ice bath. DCC (2.01 g, 9.74 mmol) was added. The ice bath wasremoved after 2.5 h and stirring was continued for another 1.5 hour atambient temperature. The precipitated DCU was removed by filtration andwashed once with DMF (15 mL), and twice with methylene chloride (2×15mL). To the combined filtrate was added more methylene chloride (100mL). The solution was washed successively with dilute sodium hydrogencarbonate (2×100 mL), dilute potassium hydrogen suite (2×100 mL), andsaturated sodium chloride (1×100 mL). The organic phase was evaporatedto dryness, in vacuo, which afforded 3.28 g (73%) of a yellowish oilysubstance. HPLC of the raw product showed a purity of only 66% withseveral impurities, both more and less polar than the main peak. The oilwas dissolved in absolute ethanol (50 mL) and activated carbon wasadded. After stirring for 5 minutes, the solution was filtered. Thefiltrate was mixed with water (30 mL) and was allowed to stir overnight.The next day, the white precipitate was removed by filtration, washedwith water, and dried, affording 1.16 g (26%) of a material with apurity higher than 98% by HPLC. Addition of water to the mother liquorafforded another 0.53 g of the product with a purity of approx. 95%.Anal. for C₂₆H₃₃N₇O₇ _(^(o)) H₂O found(calc.) C: 55.01(54.44; H:6.85(6.15) and N: 16.47(17.09). ¹H—NMR (250 MHZ, CDCl₃) 8.74 (s, 1H, AdeH-2); 8.18 (b. s, 1H, ZNH); 8.10 & 8.04 (s, 1H, H-8); 7.46-7.34 (m, 5H,Ph); 5.63 (unres. t, 1H, BOC—NH); 5.30 (s, 2H, PhCH₂); 5.16 & 5.00 (s,2H, CH ₂CON); 4.29 & 4.06 (s, 2H, CH ₂CO₂H); 4.20 (q, 2H, OCH ₂CH₃);3.67-3.29 (m, 4H, CH ₂CH ₂); 1.42 (s, 9H, t-Bu) and 1.27 (t, 3H, OCH₂CH₂). The spectrum shows traces of ethanol and DCU.

EXAMPLE 11 Synthesis of N⁶-Benzyloxycarbonyl-1-(BOC-aeg)Adenine

[0107] N⁶-Benzyloxycarbonyl-1-(BOC-aeg)adenine ethyl ester (1.48 g, 2.66mmol) was suspended in THF (13 mL) and the mixture was cooled to 0° C.Lithium hydroxide (8 mL, 1 N) was added. After 15 minutes of stirring,the reaction mixture was filtered, extra water (25 mL) was added, andthe solution was washed with methylene chloride (2×25 mL). The pH of theaqueous solution was adjusted to 2 with 1 N HCl. The precipitate wasisolated by filtration, washed with water, and dried, affording 0.82 g(58%) of the product. The product was additionally precipitated twicefrom methylene chloride/petroleum ether. Yield (after drying): 0.77 g(55%). M.p. 119° C. (decomp.). Anal. for C₂₄H₂₉N₇O₇ _(^(o)) H₂Ofound(calc.) C: 53.32(52.84); H: 5.71(5.73); N: 17.68(17.97). FAB-MS.528.5 (MH+). ¹H—NMR (250 MHZ, DMSO-d₆). 12.75 (very b, 1H, CO₂H); 10.65(bs, 1H, ZNH); 8.59 (d, 1H, J=2.14 Hz, Ade H-2); 8.31 (s, 1H, Ade H-8);7.49-7.31 (m, 5H, Ph); 7.03 & 6.75 (unresol. t, 1H, BOC—NH); 5.33 & 5.16(s, 2H, CH₂CON); 5.22 (s, 2H, PhCH ₂); 4.34-3.99 (s, 2H, CH₂CO₂H);3.54-3.03 (multiplets, includes water, CH ₂CH ₂) and 1.39 & 1.37 (s, 9H,t-Bu). ¹³C—NMR 170.4; 166.6; 152.3; 151.5; 149.5; 145.2; 128.5; 128.0;127.9; 66.32; 47.63; 47.03; 43.87 and 28.24.

EXAMPLE 12 Synthesis of 2-Amino-6-Chloro-9-Carboxymethylpurine

[0108] To a suspension of 2-amino-6-chloropurine (5.02 g, 29.6 mmol) andpotassium carbonate (12.91 g, 93.5 mmol) in DMF (50 mL) was addedbromoacetic acid (4.7 g, 22.8 mmol). The mixture was stirred vigorouslyfor 20 h under nitrogen. Water (150 mL) was added and the solution wasfiltered through celite to give a clear yellow solution. The solutionwas acidified to a pH of 3 with 4 N hydrochloric acid. The precipitatewas filtered and dried, in vacuo, over sicapent. Yield: 3.02 g (44.8%).¹H—NMR(DMSO-d₆) δ: 4.88 ppm (s, 2H); 6.95 (s, 2H); 8.10 (s, 1H).

EXAMPLE 13 Synthesis of 2-Amino-6-Benzyloxy-9-Carboxymethylpurine

[0109] Sodium (2 g 87 mmol) was dissolved in benzyl alcohol (20 mL) andheated to 130° C. for 2 h. After cooling to 0° C., a solution of2-amino-6-chloro-9-carboxymethylpurine (4.05 g, 18 mmol) in DMF (85 mL)was slowly added, and the resulting suspension stirred overnight at 20°C. Sodium hydroxide solution (1 N, 100 mL) was added and the clearsolution was washed with ethyl acetate (3×100 mL). The water phase wasthen acidified to a pH of 3 with 4 N hydrochloric acid. The precipitatewas taken up in ethyl acetate (200 mL), and the water phase wasextracted with ethyl acetate (2×100 mL). The combined organic phaseswere washed with saturated sodium chloride solution (2×75 mL), driedwith anhydrous sodium sulfate, and evaporated to dryness, in vacuo. Theresidue was recrystallized from ethanol (300 mL). Yield, after drying invacuo, over sicapent: 2.76 g (52%). M.p. 159-65° C. Anal. (calc.;found): C(56.18; 55.97), H(4.38; 4.32), N(23.4; 23.10). ¹H—NMR (DMSO-d₆)δ: 4.82 (s, 2H); 5.51 (s, 2H); 6.45 (s, 2H); 7.45 (m, 5H); 7.82 (s, 1H).

EXAMPLE 14 Synthesis ofN-([2-Amino-6-Benzyloxy-Purine-9-yl]-Acetyl)-N-(2-BOC-Aminoethyl)Glycine[BOC-Gaeg-OH Monomer]

[0110] 2-Amino-6-benzyloxy-9-carboxymethyl-purine (0.5 g, 1.67 mmol),methyl-N(2-[tert-butoxycarbonylamino]ethyl)glycinate (0.65 g, 2.8 mmol),diisopropylethyl amine (0.54 g, 4.19 mmol), andbromo-tris-pyrrolidino-phosphonium-hexafluoro-phosphate (PyBroP®) (0.798g, 1.71 mmol) were stirred in DMF (2 mL) for 4 h. The clear solution waspoured into an ice-cooled solution of sodium hydrogen carbonate (1 N, 40mL) and extracted with ethyl acetate (3×40 mL). The organic layer waswashed with potassium hydrogen sulfate solution (1 N, 2×40 mL), sodiumhydrogen carbonate (1 N, 1×40 mL) and saturated sodium chloride solution(60 mL). After drying with anhydrous sodium sulfate and evaporation invacuo, the solid residue was recrystallized from 2:1 ethylacetate/hexane (20 mL) to give the methyl ester in 63% yield. (MS-FAB514 (M+1). Hydrolysis was accomplished by dissolving the ester in 1:2ethanol/water (30 mL) containing concentrated sodium hydroxide (1 mL).After stirring for 2 h, the solution was filtered and acidified to a pHof 3, by the addition of 4 N hydrochloric acid. The title compound wasobtained by filtration. Yield: 370 mg (72% for the hydrolysis). Purityby HPLC was more than 99%. Due to the limited rotation around thesecondary amide several of the signals were doubled in the ratio 2:1(indicated in the list by mj for major and mi for minor). ¹H—NMR(250.MHZ, DMSO-d₆) δ: 1.4 (s, 9H); 3.2 (m, 2H); 3.6 (m, 2H); 4.1 (s. mj,CONRCH ₂COOH); 4.4 (s, mi, CONRCH ₂COOH); 5.0 (s, mi, Gua-CH ₂CO—); 5.2(s, mj, Gua-CH ₂CO); 5.6 (s, 2H); 6.5 (s, 2H); 6.9 (m, mi, BOC—NH); 7.1(m, mj, BOC—NH); 7.5 (m, 3H); 7.8 (s, 1H); 12,8 (s, 1H). ¹³C—NMR.170.95; 170.52; 167.29; 166.85; 160.03; 159.78; 155.84; 154.87; 140.63;136.76; 128.49; 128.10; 113.04; 78.19; 77.86; 66.95; 49.22; 47.70;46.94; 45.96; 43.62; 43.31 and 28.25.

EXAMPLE 15 Synthesis ofEthyl-N⁶-(Benzyloxycarbonyl)-2,6-Diaminopurin-9-yl-Acetate

[0111] To a suspension of 2,6-diaminopurine (3 g, 19.46 mmol) in dry DMF(90 mL) was added NaH (60% in oil, 0.87 g, 21.75 mmol). After 1 hourethyl bromoacetate (4.23 g, 25.34 mmol) was added. The reaction mixturebecame homogenous in 30 minutes and was allowed to stir for anadditional 90 minutes. The DMF was removed in vacuo resulting in a tanpowder. The tan powder was then refluxed with 1,4-dioxane (200 mL) for10 minutes and filtered through celite. The solution was concentrated togive a light yellow powder. To the light yellow powder (5.52 g) in1,4-dioxane (150 mL) was added freshly preparedN-benzyloxycarbonyl-N′-methylimidazolium triflate (10.7 g, 29.2 mmol).The reaction mixture was stirred at room temperature for 16 h resultingin a reddish solution. The dioxane was removed in vacuo and the crudematerial was recrystallized from MeOH:diethyl ether to give 4.56 g (63%)of the title compound as a cream-colored solid.

[0112]¹H NMR (DMSO-d₆) δ: 10.12 (bs, 1H), 7.43 (m, 5H), 6.40 (bs, 2H),5.17 (s, 2H), 4.94 (s, 2H), 4.18 (q, J=7.2, 3H), 1.21 (t, J=7.2, 3H).¹³C NMR (DMSO-d₆) ppm: 167.81, 159.85, 154.09, 152.07, 149.77, 140.62,136.42, 128.22, 127.74, 127.61, 166.71. 65.87, 61.21, 43.51, 13.91.

EXAMPLE 16 Synthesis ofN⁶-(Benzyloxycarbonyl)-2,6-Diaminopurin-9-yl-Acetic Acid

[0113] Ethyl-N⁶-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetate (3 g,8.1 mmol) was dissolved in NaOH (2 N, 30 mL). After 1 h the solution wasacidified to pH 2.5 with 2 M HCl. The precipitate was filtered, washedwith water, and dried to give 2.82 g (98%) of the title compound as awhite solid.

[0114] IR (KBr): 3300, 3095, 1750, 1630, 1590, 1410. ¹H NMR(DMSO-d₆) δ:10.11 (s, 1H), 7.91 (s, 1H), 7.45-7.33 (m, 5H), 6.40 (s, 2H), 5.17 (s,2H), 4.83 (s, 2H).

EXAMPLE 17 Synthesis of BOC-Aminoacetaldehyde

[0115] The title compound was prepared according to a publishedliterature procedure (Dueholm et al., Organic Preparations andProcedures Intl., 1993, 25, 457).

EXAMPLE 18 Synthesis of ε-(2-Chlorobenzyloxycarbonyl)-Lysine Allyl Ester

[0116] The title compound was prepared according to a publishedliterature procedure (Waldmann and Horst, Liebigs Ann. Chem, 1983,1712).

EXAMPLE 19 Synthesis ofN-(BOC-Aminoethyl)-ε-(2-Chlorobenzyloxycarbonyl)-Lysine Allyl Ester

[0117] ε-(2-chlorobenzyloxycarbonyl)-lysine allyl ester (from example18) was dissolved in methanol (50 mL) and cooled to 0° C. To theresulting solution was added sodium cyanoborohydride (5.9 mmol) followedby acetic acid (0.75 mL). After 5 minutes BOC-amino-acetaldehyde (13.3mmol) was added and the reaction mixture was stirred for an additional 1h. The methanol was removed in vacuo and the oil was dissolved in ethylacetate (40 mL), washed with saturated aqueous NaHCO₃, brine, dried overNa₂SO₄ and concentrated to give a clear colorless oil. This oil wasdissolved in dry ether (80 mL), cooled to −20° C., and a molarequivalent of HCl in ether was added slowly. The resulting white solidwas collected by filtration and air dried. Precipitation of theair-dried white solid from dry ether gave analytically pure titlecompound.

EXAMPLE 20 Synthesis ofN-(BOC-Aminoethyl)-N-[N′(Benzyloxycarbonyl)2,6-Diaminopurin-9-yl-Acetyl]-ε-(2-Chlorobenzyloxycarbonyl)-LysineAllyl Ester

[0118] To N⁶-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetic acid (3.6g, 10.5 mmol) in DMF (150 mL) was added N,N-diisopropylethylamine (2.75mL, 21 mmole), andN-(BOC-aminoethyl)-ε-(2-chlorobenzyloxycarbonyl)-lysine allyl esterhydrochloride (7.31 gm, 15.8 mmol). The reaction mixture was stirredunder nitrogen for 20 minutes and bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBrop, 5.4 gm, 11.6 mmol) was added. The reactionmixture was stirred overnight at room temperature under an atmosphere ofnitrogen gas. The resulting mixture was concentrated and dissolved inethyl acetate. The ethyl acetate solution was washed with aqueoussaturated sodium bicarbonate, separated and concentrated. The crudematerial was purified by silica gel flash column chromatography usingethyl acetate:hexane: methanol (6:3:1, v/v/v), as the eluent.Concentration and drying of the appropriate fractions gave 3.1 g (37%)of the title compound.

EXAMPLE 21 Synthesis ofN-(BOC-Aminoethyl)-N-[N⁶-(Benzyloxycarbonyl)-2,6-Diaminopurin-9-yl-Acetyl]-ε-(2-Chlorobenzyloxycarbonyl)-Lysine

[0119] ToN-(BOC-aminoethyl)-N-[N⁶-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetyl]-ε-(2-chlorobenzyloxycarbonyl)-lysineallyl ester hydrochloride (3.1 gm, 3.93 mmol) was added THF (100 mL)morpholine (3.5 mL, 39.3 mmol), andtetrakis(triphenylphosphine)-palladium(0) (0.45 gm, 0.393 mmol). Thereaction mixture was stirred under an atmosphere of nitrogen for 2.5 hat room temperature. The resulting mixture was concentrated anddissolved in ethyl acetate. The ethyl acetate solution was washed withaqueous saturated potassium hydrogen sulfate (that was half-diluted withwater), separated and concentrated. The crude material was purified bysilica gel flash column chromatography using chloroform:methanol (9:1,v/v), as the eluent. Concentration and drying of the appropriatefractions gave 1.25 g (42%) of the title compound.

EXAMPLE 22 Preparation of Guanine Monomer (BOC-Gaek-OH)

[0120] To N⁶-benzyl-9-carboxymethylene-guanine (2.63 g, 8.78 mmol) wasadded DIEA (2.6 mL, 20 mmol), DMF (30 mL), dichloromethane (70 mL), andN-(BOC-aminoethyl)-ε-(2-chlorobenzyloxycarbonyl)-lysine allyl ester (3.7g, 8.04 mmol). The reaction mixture was stirred under nitrogen for 20minutes. PyBrop (4 g, 8.58 mmol) was added and the reaction mixturestirred for an additional 16 h. The reaction mixture was concentratedand the residue was purified by silica gel flash column chromatographyusing chloroform/hexanes/methanol (12:7:1, v/v/v) to give 4 g (60%) ofthe title compound as the allyl ester.

[0121] To the allyl ester (4 g, 5.37 mmol) was added THF (100 mL),tetrakispalladium(0) (0.18 g, 0.15 mmol), and morpholine (6.1 mL, 70mmol). The reaction mixture was stirred under nitrogen for 2.5 h andconcentrated. The residue was purified by silica gel flash columnchromatography using chloroform/hexanes/methanol (11:8:1, v/v/v) to give2.67 g (60%) of the title compound.

EXAMPLE 23 Preparation of Adenine Monomer (BOC-Aaek-OH)

[0122] The procedure used for the guanine monomer in Example 22 abovewas followed for the synthesis of the adenine monomer usingN6-benzyl-9-carboxymethylene-adenine.

EXAMPLE 24 Preparation of Cytosine Monomer (BOC-Caek-OH).

[0123] To N-(BOC-aminoethyl)-ε-(2-chlorobenzyloxycarbonyl)-lysine allylester (8.21 g, 17.7 mmol), were added triethylamine (10 mL, 98 mmol) anddichloromethane (200 mL). The solution was cooled to about 0° C. in anice bath under nitrogen. To the cooled solution was added chloroacetylchloride (2.2 mL, 27.6 mmol) over 10 minutes and the reaction mixturestirred at room temperature for 16 h. The reaction mixture wasconcentrated and the residue was purified by silica gel flash columnchromatography using ethyl acetate/hexanes (1:1, v/v) to give 6.54 g(68%) of the N-acetylated lysine backbone.

[0124] Cytosine was protected at the N⁴-position by treatment withbenzyl chloroformate in pyridine at 0° C. to give N⁴-benzyl-cytosine.

[0125] To N4benzyl-cytosine (1.31 g, 5.34 mmol) was added DMF (200 mL),and 60% NaH in mineral oil (0.22 g, 5.4 mmol) and the resulting mixturewas stirred under nitrogen for 30 minutes. To the resulting mixture wasadded the N-acetylated lysine backbone (2.9 g, 5.34 mmol) in DMF (25 mL)and the mixture stirred for 16 h. The reaction mixture was concentratedand the residue dissolved in dichloromethane (250 mL). Thedichloromethane phase was washed with water (200 mL) and concentrated.The resulting residue was purified by silica gel flash columnchromatography using dichloromethane:hexanes:methanol (8:2:1) to give2.4 g (85%) of the cytosine attached to the aminoethyl-lysine backboneas the allyl ester.

[0126] The allyl ester is converted to the active monomer bydeprotection using palladiun following the procedure used in Example 22above to give 1.05 g (46%) of the title compound.

EXAMPLE 25 Preparation of Thymine Monomer (BOC-Taek-OH)

[0127] The thymine monomer was prepared following the procedure ofExample 24 above.

EXAMPLE 26 Solid Phase Synthesis of H-Taeg-Aaeg-[Taeg]₈-Lys-NH₂

[0128] (a) Stepwise Assembly of BOC-Taeg-A(Z)aeg-[Taeg]₈-Lys(CIZ)-MBHAResin.

[0129] About 0.3 g of wet BOC-[Taeg]₈-Lys(CIZ)-MBHA resin was placed ina 3 mL SPPS reaction vessel. BOC-Taeg-A(Z)aeg-[Taeg]₈-Lys(CIZ)-MBHAresin was assembled by in situ DCC coupling (single) of the A(Z)aegresidue utilizing 0.19 M of BOC-A(Z)aeg-OH together with 0.15 M DCC in2.5 mL of 50% DMF/CH₂Cl₂ and a single coupling with 0.15 M BOC-Taeg-OPfpin neat CH₂Cl₂ (“Synthetic Protocol I”). The synthesis was monitored bythe quantitative ninhydrin reaction, which showed about 50%incorporation of A(Z)aeg and about 96% incorporation of Taeg.

[0130] (b) Cleavage, Purification, and Identification ofH-Taeg-Aaeg-[Taeg]₈-Lys-NH₂.

[0131] The protected BOC-Taeg-A(Z)aeg-[Taeg]₈-Lys(CIZ)-BHA resin wastreated with 50% trifluoroacetic acid in methylene chloride to removethe N-terminal BOC group (which is a precursor of the potentiallyharmful tert-butyl cation) prior to the HF cleavage. Followingneutralization and washing (performed in a way similar to those of steps2-4 in “Synthetic Protocol 1”), and drying for 2 h in vacuum, theresulting 53.1 mg of H-[Taeg]₅-BHA resin was cleaved with 5 mL ofHF:anisole (9:1, v/v) while stirring at 0° C. for 60 minutes. Afterremoval of HF, the residue was stirred with dry diethyl ether (4×15 mL,15 minutes each) to remove anisole, filtered under gravity through afritted glass funnel, and dried. The PNA was then extracted into a 60 mL(4×15 mL, stirring 15 minutes each) 10% aqueous acetic acid solution.Aliquots of this solution were analyzed by analytical reverse-phase HPLCto establish the purity of the crude PNA. The main peak at 13 minutesaccounted for about 93% of the total absorbance. The remaining solutionwas frozen and lyophilized to afford about 15.6 mg of crude material.The main peak at 14.4 minutes accounted for less than 50% of the totalabsorbance. A 0.5 mg portion of the crude product was purified to giveapproximately 0.1 mg of H-Taeg-Aaeg-[Taeg]₈-Lys-NH₂. For (MH+)⁺ thecalculated m/z value was 2816.16 and the measured m/z value was 2816.28.

[0132] (c) Synthetic Protocol I.

[0133] (1) BOC-deprotection with TFA/CH₂Cl₂ (1:1, v/v), 2.5 mL, 3×1minute and 1×30 minutes; (2) washing with CH₂Cl₂, 2.5 mL, 6×1 minute;(3) neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 2.5 mL, 3×2 minutes;(4) washing with CH₂Cl₂, 2.5 mL, 6×1 minute, and drain for 1 minute; (5)2-5 mg sample of PNA-resin was removed and dried thoroughly for aquantitative ninhydrin analysis to determine the substitution; (6)addition of 0.47 mmol (0.25 g) BOC-A(Z)aeg-OH dissolved in 1.25 mL ofDMF followed by addition of 0.47 mmol (0.1 g) DCC in 1.25 mL of CH₂Cl₂or 0.36 mmol (0.2 g) BOC-Taeg-OPfp in 2.5 mL of CH₂Cl₂; the couplingreaction was allowed to proceed for a total of 20-24 h while shaking;(7) washing with DMF, 2.5 mL, 1×2 minutes; (8) washing with CH₂Cl₂, 2.5mL, 4×1 minute; (9) neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 2.5 mL,2×2 minutes; (10) washing with CH₂Cl₂, 2.5 mL, 6×1 minute; (11) 2-5 mgsample of protected PNA-resin was removed and dried thoroughly for aquantitative ninhydrin analysis to determine the extent of coupling;(12) blocking of unreacted amino groups by acetylation with a 25 mLmixture of acetic anhydride/pyridine/CH₂Cl₂ (1:1:2, v/v/v) for 2 h(except after the last cycle); and (13) washing with CH₂Cl₂, 2.5 mL, 6×1minute; (14) 2×2-5 mg samples of protected PNA-resin are removed,neutralized with DIEA/CH₂Cl₂ (1:19, v/v) and washed with CH₂Cl₂ forninhydrin analyses.

EXAMPLE 27 Solid Phase Synthesis of H-[Taeg]₂-Aaeg-[Taeg]₅-Lys-NH₂

[0134] (a) Stepwise Assembly ofBOC-[Taeg]₂-A(Z)aeg-[Taeg]₅-Lys(CIZ)-MBHA Resin.

[0135] About 0.5 g of wet BOC-[Taeg]₅-Lys(CIZ)-MBHA resin was placed ina 5 mL SPPS reaction vessel. BOC-[Taeg]₂-A(Z)aeg-[Taeg]₅-Lys(CIZ)-MBHAresin was assembled by in situ DCC coupling of both the A(Z)aeg and theTaeg residues utilising 0.15 M to 0.2 M of protected PNA monomer (freeacid) together with an equivalent amount of DCC in 2 mL neat CH₂Cl₂(“Synthetic Protocol II”). The synthesis was monitored by thequantitative ninhydrin reaction which showed a total of about 82%incorporation of A(Z)aeg after coupling three times (the first couplinggave about 50% incorporation; a fourth HOBt-mediated coupling in 50%DMF/CH₂Cl₂ did not increase the total coupling yield significantly) andquantitative incorporation (single couplings) of the Taeg residues.

[0136] (b) Cleavage, Purification, and Identification ofH-[Taeg]₂-Aaeg-[Taeg]₅-Lys-NH₂.

[0137] The protested BOC-[Taeg]₂-A(Z)aeg-[Taeg]-Lys(CIZ)-BHA resin wastreated as described in Example 26(b) to yield about 16.2 mg of crudematerial upon HF cleavage of 102.5 mg dryH-[Taeg]₂-A(Z)aeg-[Taeg]₅-Lys(CIZ)-BHA resin. A small portion of thecrude product was purified. For (MH+)⁺, the calculated m/z value was2050.85 and the measured m/z value was 2050.90

[0138] (c) Synthetic Protocol II.

[0139] (1) BOC-deprotection with TFA/CH₂Cl₂ (1:1, v/v), 2 mL, 3×1 minuteand 1×30 minutes; (2) washing with CH₂Cl₂, 2 mL, 6×1 minute; (3)neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 2 mL, 3×2 minutes; (4)washing with CH₂Cl₂, 2 mL, 6×1 minute, and drain for 1 minute; (5) 2-5mg sample of PNA-resin was removed and dried thoroughly for aquantitative ninhydrin analysis to determine the substitution; (6)addition of 0.44 mmol (0.23 g) BOC-A(Z)aeg-OH dissolved in 1.5 mL ofCH₂Cl₂ followed by addition of 0.44 mmol (0.09 g) DCC in 0.5 mL ofCH₂Cl₂ or 0.33 mmol (0.13 g) BOC-Taeg-OH in 1.5 mL of CH₂Cl₂ followed byaddition of 0.33 mmol (0.07 g) DCC in 0.5 mL of CH₂Cl_(2,); the couplingreaction was allowed to proceed for a total of 20-24 h with shaking; (7)washing with DMF, 2 mL, 1×2 minutes; (8) washing with CH₂Cl₂, 2 mL, 4×1minute; (9) neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 2 mL, 2×2minutes; (10) washing with CH₂Cl₂, 2 mL, 6×1 minute; (11) 2-5 mg sampleof protected PNA-resin was removed and dried thoroughly for aquantitative ninhydrin analysis to determine the extent of coupling;(12) blocking of unreacted amino groups by acetylation with a 25 mLmixture of acetic anhydride/pyridine/CH₂Cl₂ (1:1:2, v/v/v) for 2 h(except after the last cycle); (13) washing with CH₂Cl₂, 2 mL, 6×1minute; and (14) 2×2-5 mg samples of protected PNA-resin were removed,neutralized with DIEA/CH₂Cl₂ (1:19, v/v) and washed with CH₂Cl₂ forninhydrin analyses.

EXAMPLE 28 Standard Protocol for PNA Synthesis and Characterization

[0140] Instrument: PerSeptive Biosystems 8909 Expedite.

[0141] Synthesis Scale: 2 μmole.

[0142] Reagents:

[0143] Wash A: 20% DMSO in NMP

[0144] Wash B: 2 M Collidine in 20% DMSO in NMP

[0145] Deblock: 5% m-Cresol, 95% TFA

[0146] Neutralizer: 1 M DIEA in 20% DMSO in NMP

[0147] Cap: 0.5 M Acetic Anhydride, 1.5 M Collidine in 20% DMSO in NMP

[0148] Activator: 0.2 M HATU in DMF

[0149] Monomers: 0.22 M in 2 M Collidine (50% Pyridine in DMF)

[0150] Synthesis: The solid support (BOC-BHA-PEG-resin) is washed with708 μl of Wash A. Deblock (177 μL) is passed through the column 3 timesover 6.3 minutes. The resin is then washed with 1416 μL of Wash A. Thefree amine is neutralized with 1063 μL of Neutralizer. The resin iswashed with 1062 L of Wash B. Monomer and Activator (141 μL each) areslowly added to the column over 14 minutes. The resin is washed with 708μL of Wash B and 708 μL of Wash A. Unreacted amine is capped with slowaddition of 708 μL of Cap solution over 5 minutes. The resin is thenwashed 2124 μL of Wash A. The cycle is repeated until synthesis of thedesired PNA sequence is completed.

[0151] Cleavage: The PNA-resin is washed with 5 mL of MeOH and driedunder vacuum. The dried resin is emptied into a 1.5 mL Duraporeultrafree filter unit. Thioanisole (25 μL), 25 μL of m-Cresol, 100 μL ofTFA and 100 μL of TFMSA is added to the resin, vortexed for about 30seconds and allowed to stand for 2 h. The reaction mixture is thencentrifuged for 5 minutes at 10 K and the inner tube with resin isremoved. Approximately 1.5 mL of ether is added to the TFA solution toprecipitate the product. The TFA solution is vortexed, followed bycentrifugation at 10 K for 2 minutes. The ether is removed in vacuo.Ether precipitation and centrifugation are repeated an additional 2times. The dry pellet is heated in a heat block (55° C.) for 15 to 30minutes to remove excess ether and redissolved in 200 μL of H₂O. Solventis added to 100 mg of Dowex Acetate Resin in a 1.5 mL Durapore ultrafreefilter unit, vortexed, allowed to stand for 30 minutes and centrifugedat 10 K for 2 minutes.

[0152] Characterization: The absorbance of a 1 μL sample in 1 mL of H₂Ois measured at 260 nm. Isopropanol (50%) in H₂O with 1% Acetic acid (100μL) is added to 4 μL of the sample. This sample is characterized byelectrospray mass spectrometry. Common Abreviations NMP: N-methylpyrrolidinone TFA: Trifluoroacetic acid DIEA: N,N-DiisopropylethylamineHATU: O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate TFMSA: Trifluormethanesulfonic Acid

EXAMPLE 29 Synthesis and Cellular Uptake of Conjugated PNA Oligomers

[0153] Using the procedures of Example 28, the aminoethylglycine PNAmonomers of examples 5 through 14, and monomers of Examples 15 through21, the following PNA oligomers were synthesized. PNA Liposomes CellularUptake Fl-GGT-GCT-CAC-GCT-GGC-Lys-NH2 x − Fl-GGT-GCT-CAC-GCT-GGC-Lys-NH2✓ − Ada-Fl-GGT-GCT-CAC-TGC-GGC-Lys-NH2 x −Ada-Fl-GGT-GCT-CAC-TGC-GGC-Lys-NH2 ✓ (+)Fl-GGTk-GCTk-CAC-TkGC-GGC-Lys-NH2 x − Fl-GGTk-GCTk-CAC-TkGC-GGC-Lys-NH2✓ − Ada-Fl-GGTk-GCTk-CAC-TkGC-GGC-Lys-NH2 x +Ada-Fl-GGTk-GCTk-CAC-TkGC-GGC-Lys-NH2 ✓ +

EXAMPLE 30 Synthesis of Linolenyl-TAG-CAG-AGG-AGC-TC (SEQ ID NO:1)

[0154] Linolenic acid (40 μmoles) was dissolved in coupling solvent (100μL) (0.5 M DIEA in 20% DMSO/NMP),to which HATU (90 μL of 0.4 M) wasadded and the solution was mixed. After a 2 minute activation period,the solution was mixed with protected PNA resin (15.4 mg, 2 μmoles).After 1 hour, the resin was washed with 20% DMSO/NMP, CH₂Cl₂ and MeOH(about 3 mL each). The resulting linolenyl-conjugated PNA was cleavedfrom the solid support and characterized according to the proceduredescribed in Example 28.

EXAMPLE 31 Synthesis of Oleyl-TAG-CAG-AGG-AGC-TC (SEQ ID NO:1)

[0155] Oleic acid (40 μmoles) was dissolved in coupling solvent (100 μL) (0.5 M DIEA in 20% DMSO/NMP), to which HATU (90 μL of 0.4 M was addedand the solution was mixed. After a 2 minute activation period, thesolution was mixed with protected PNA resin (5.4 mg, 2 μmoles). After 1hour, the resin was washed with 20% DMSO/NMP, CH₂Cl₂ and MeOH (about 3mL each). The resulting oleyl-conjugated PNA was cleaved from the solidsupport and characterized according to the procedure described inExample 28.

EXAMPLE 32 Synthesis of Caproyl-Gly-TAG-CAG-AGG-AGC-TC (SEQ ID NO:1)

[0156] Caproyl-gly (40 μmoles) was dissolved in coupling solvent (100μL) (0.5 M DIEA in 20% DMSO/NMP), to which HATU (90 μL of 0.4 M) wasadded and the solution was mixed. After 2 minutes of activation, thesolution was mixed with protected PNA resin (15.4 mg, 2 μmoles). After 1hour, the resin was washed with 20% DMSO/NMP, CH₂Cl₂ and MeOH (about 3mL each). The resulting PNA was cleaved from the solid support andcharacterized according to the procedure described in Example 28.

EXAMPLE 33 Synthesis of N-BOC-ε-Fluoresceinyl Carbonyl)-D-Lysine and itsEthyl Ester

[0157] α-BOC protected lysine ethyl ester was treated with excessfluorescein isocyanate in a mixture of THF and DMF at room temperaturefor several hours. The reaction was monitored by tlc for thedisappearance of the starting amino acid. The reaction was then treatedwith equal volumes of water and chloroform and the phases separated. Theaqueous phase was extracted with more chloroform and the combinedorganic solutions so obtained, dried with magnesium sulfate. Thissolution was concentrated, in vacuo, and the crude product obtained waspurified by column chromatography to afford the N-BOC-ε-(Fluoresceinylcarbonyl)-D-lysine ethyl ester.

[0158] The ethyl ester was hydrolyzed using 1M aqueous lithium hydroxideand tetrahydrofuran as solvent. The progress of the reaction wasfollowed by tlc and upon completion the reaction mixture was treatedwith water and then washed 2× with dichloromethane. The basic solutionwas then cooled to <10 C., neutralized with 1N HCl to a pH below 4 andthe product extracted out using ethyl acetate. The organic extract wasdried using magnesium sulfate, and concentrated in vacuo to afford theN-BOC-ε-(Fluoresceinyl carbonyl)-D-lysine.

EXAMPLE 34 Coupling of N-BOC-e-(Fluoresceinyl Carbonyl)-D-Lysine to PNAof Sequence GGT-GCT-CAC-TGC-GGC-Lys-NH₂ (SEQ ID NO:2)

[0159] PNA of sequence GGT-GCT-CAC-TGC-GGC-Lys-NH₂ was synthesizedfollowing standard PNA synthesis protocols (as in examples 27 and 28)and commencing with lysine-derivatized synthesis resin. The N-terminalBOC group of the PNA bound to resin was deprotected using:

[0160] 1. 3 mL of 1:1 v/v TFA/DCM 1×2 mins and then 1×0.5 hours

[0161] 2. Washing with 3 mL DCM, 4×20 seconds.

[0162] Washing with 3 mL DMF, 2×20 seconds

[0163] Washing with 3 mL DCM, 2×20 seconds.

[0164] Draining for 30 seconds.

[0165] 3. Neutralizing with 3 mL DIEA/DCM, 1:19 v/v, 2×3 minutes.

[0166] Coupling of the Fluoresceinyl lysine was then performed accordingto the following steps:

[0167] 1. Wash with DCM, 3 mL, 4×20 seconds.

[0168] Drain for 1 minute.

[0169] 2. Addition of 4 equivalents of DIC, and 4 equivalents ofN-BOC-e-(Fluoresceinyl carbonyl)D-Lysine dissolved in 1:1 v/v DCM/DMF(final concentration of the amino acid being 0.1M).

[0170] 3. Coupling allowed to proceed for 0.5 hour with shaking at roomtemperature.

[0171] 4. Drain for 20 seconds.

[0172] Wash with 3 mL DMF, 2×20 seconds and 1×2 minutes.

[0173] Wash with 3 mL DCM, 4×20 seconds.

[0174] 5. Neutralize with 3 mL, DIEA/DCM, 1:19 v/v, 2×3 minutes.

[0175] Wash with 3 mL DCM, 4×20 seconds; Drain for 1 minute.

[0176] 6. Perform a qualitative Kaiser test. A negative result indicatesnear 100% coupling.

[0177] The BOC group was cleaved and then the PNA(Fl-GGT-GCT-CAC-TGC-GGC-Lys-NH₂) was cleaved and purified as in examples27 and 28. Alternatively, the PNA is left attached to the resin and usedfor derivatization with a lipophilic group as in example 35.

EXAMPLE 35 Synthesis of Ada-Fl-GGT-GCT-CAC-TGC-GGC-Lys-NH₂ (SEQ ID NO:2)

[0178] The N-terminal BOC group of Fl-GGT-GCT-CAC-TGC-GGC-Lys-NH₂ PNAbound to the resin was first cleaved (as in example 34) and the freeamino terminus then derivatized as follows:

[0179] 100 μmole adamantoyl chloride was dissolved in 1:5 v/v DIEA/DMFwas added to 2 μmole resin bound PNA (that is completely protectedexcept at the N-terminus where the BOC group has been cleaved). After 1hour of reaction, the resin was washed with 3 mL each of 20% NMP/DMSO,DCM and methanol. The PNA was cleaved from the resin and purifiedfollowing standard protocols as in examples 27 and 28.

EXAMPLE 36 Synthesis of Adamantyl-Ahx-TAG-CAG-AGG-AGC-TC (SEQ ID NO:1)

[0180] Adamantyl carbonyl chloride (100 μmoles) was dissolved in DMF(1.0 mL) and DIEA (200 μmoles). This solution was mixed with protectedPNA resin (15.4 mg, 2 μmoles) with an attached amino hexanoic acid grouplinking group. After 1 hour, the resin was washed with 20% DMSO/NMP,CH₂Cl₂ and MeOH (about 3 mL each). The resulting PNA was cleaved fromthe solid support following known methods and techniques.

EXAMPLE 37 Preparation of PNA/Liposome

[0181] Liposomes containing the PNA Adamantyl-(Fl*)-TTT AGC TTCAGC-LysNH₂ (SEQ ID NO:3), where Fl* is a fluoresceinated PNA monomer,were prepared by a modification of the ethanol injection methoddescribed by Campbell. Biotechniques, 1995, 18, 1027. DOPE(dioleyl-L-a-phosphatidylethanolamine) (13.4 mmol) and DDAB(dimethyldiocadecylammonium bromide) (6.6 mmol) were dissolved in 1 mLof 96% ethanol. A solution of PNA (10 mL, 2.5 mM) in DMSO was combinedwith the lipid mixture (40 mL). The resulting 50 mL of material was thenrapidly added to sterile distilled H₂O (1 mL) while vortexing. Theresulting PNA concentration in the liposome mix was 25 mM. For celluptake experiments, the PNA-liposome mix (40 mL) was added to OptiMEM™(1 mL) and fed to cells. The final concentration of PNA was 1 mM.

[0182] Liposome transfection reagents: Four commercially availabletransfection liposome reagents were employed: Lipofectin™ (Gibco BRL),Lipofectamine™ (Gibco BRL), Tfx-50™ (Promega) and DOTAP™ (BoehringerMannheim). Each liposome reagent was mixed with conjugated PNA 1118 togive a final PNA concentration of 1 mM in the culture medium (1 mL). Theoptimal concentration of each liposome reagent in terms of PNA celluptake was determined. The table below shows the amount of reagent usedper mL of OptiMEM. Lipofectin ™ Lipofectamine ™ Tfx-50 ™ DOTAP ™ 2 mL 4mL 10 mL 10 mL

[0183] Cells: The human carcinoma cell line HeLa was grown in RPMI 1640medium containing Glutamax™, penicillin, streptomycin and fetal bovineserum. On the day preceding the experiment, the cells were plated at adensity of 2×10⁵ cells per dish in 35 mm dishes containing coverslips.The following day the cells were washed once with OptiMEM, then fed with1 mL OptiMEM containing 1 μM PNA or PS-ODN, either alone, mixed with oneof the 4 liposome reagents or incorporated in DOPE/DDAB liposomes, asdescribed above. After an overnight incubation, the PNA-treated cellswere fixed in 3% formaldehyde/0.2% glutaraldehyde on ice. The coverslipswere then mounted on objective glasses and the cells observed byfluorescence microscopy on a Leits Diaplan microscope. Micrographs weretaken with Kodak Ektacrome 1600 ASA film.

EXAMPLE 38 Cellular Uptake of Conjugated PNAs

[0184] Four conjugated PNAs (Example 30-32 and 36) having the titleformula were prepared following the standard procedure illustrated inExample 28. Lysine residues were incorporated into PNA's by using amodified MBHA resin (Dueholm, J. Org. Chem., 1994, 59, 5767) using aBoc-Lys-ClCbz (ClCbz=2-chlorobenzyloxycarbonyl). The PNA oligomer wasthen extended with a protected Lys group that was previouslyfluoresceinated at the ε-amino group. Deprotection of the amino groupfollowed by conjugation with a lipophilic group afforded the supportbound conjugated PNA. Cleavage from the solid support afforded the freePNA conjugate having a fluorescent label. The lipophilic groups (R)investigated include adamantoyl, decanoyl, heptyl-succinyl andpalmityl-succinyl groups (as shown in FIG. 1).

[0185] Stock solutions of the four conjugated PNAs were prepared bydissolving the PNAs in DMSO. Dilutions of these stock solutions weremade in either water or OptiMEM (Gibco BRL). The human carcinoma cellline HeLa was grown in RPMI 1640 medium containing Glutamax™,penicillin, streptomycin and fetal calf serum (10% v/v). On the daypreceding the experiment, the cells were plated at a density of 2×10⁵cells per dish in 35 mm dishes containing coverslips. The next day thecells were rinsed once with OptiMEM, then fed with 3 μM PNA in 1 mL ofOptiMEM and further incubated. In order to visualize PNA uptake, thecoverslips were washed twice with PBS and the cells were fixed for 15minutes in 3% formaldehyde/0.2% glutaraldehyde on ice. After washingtwice with PBS, the coverslips were mounted on objective glasses using90% glycerol in PBS, and the cells were observed by fluorescentmicroscopy on a Leitz Diaplan Microscope. Micrographs were taken withKodak Ektachrome 1600 ASA film.

[0186] The four conjugated PNAs were tested for uptake into human cellsin culture. The PNAs were added directly to the cell culture medium.HeLa cells grown on coverslips were incubated with PNA (3 μm) in serumfree medium overnight, then fixed and examined by fluorescencemicroscopy. Both the palmityl-succinyl and the heptyl-succinylconjugated PNAs showed punctate and spotted fluorescence in all cells.Generally, the spots were evenly distributed over the cell with atendency of an enhanced staining at the edges of the cells, probably thecell membrane. The adamantoyl- and decanoyl-conjugated PNAs showed muchless cell-associated fluorescence with large fluorescent aggregates seenoutside the cells.

[0187] The palmityl-succinyl PNA conjugate was further studied byconfocal microscopy to determine the exact location and distribution ofthe PNA conjugate inside the cell. A cell was selected from the abovestudy and further scanned through 12 sections. The images confirm thatthe PNA conjugate was indeed taken up by the cells and distributed inspots throughout the cytoplasm. There was, apparently, no fluorescencein the nucleus. This pattern is indicative of the endocytotic pathway ofuptake, implying that the PNA conjugates end up in endosomes.

[0188] The palmityl-succinyl PNA conjugate was also observed in a timecourse experiment. Cells were incubated for different lengths of time inthe presence of 3 μM of PNA. The uptake of the PNA conjugate by thecells increased with time up until 24 hours of incubation when thePNA-containing medium was replaced with fresh serum containing medium.After 48 hours intracellular PNA was concentrated in compartments of thecells, probably secondary lysosomes. After 72 hours there was virtuallyno PNA left inside the cells.

EXAMPLE 39 In vitro Translation Assay using Conjugated PNAs

[0189] PNA having the sequenceR-Lys(Fluorescein)-TTT-AGC-TTC-CTT-AGC-Lys-NH₂ (SEQ ID NO:3) iscomplementary to the 15 nucleotides immediately 5′ to the AUG startcodon in CAT mRNA and the corresponding unconjugated PNA has previouslybeen shown to be able to inhibit translation of CAT in vitro. The fourconjugated PNAs of Example 38 (SEQ ID NO:2) were tested in this assay.All four conjugated PNAs specifically inhibited CAT translation atsimilar concentrations as the unconjugated PNA.

EXAMPLE 40 Preparation of Lysosome Constructs using PNA Conjugates (SEQID NO:2)

[0190] Lysosome constructs were prepared using two of the conjugatedPNAs having SEQ ID NO:2. The adamantoyl- and decanoyl-conjugated PNAswere combined with liposomes by a modification of the ethanol injectionmethod described by Campbell in Biotechniques, 1995, 18, 1027. Followingthis method, 13.4 μmole of DOPE (dioleyl-L-α-phosphatidylethanolamine)and 6.6 μmole of DDAB (dimethyldioctadecylammonium bromide) weredissolved in 1 mL of absolute ethanol. A solution of PNA (10 μL, 3 mMPNA/DMSO) was combined with 40 μL of the lipid mixture. The resulting 50μL of reaction mixture was then rapidly added to 1 mL of steriledistilled H₂O while vortex mixing. The PNA concentration in the liposomemixture was thus 30 μM. For cell uptake experiments, 60 μL of thePNA-liposome mixture was added to 1 mL of OptiMEM and fed to the cells.

[0191] Incorporation of the conjugated PNAs into the liposome constructswas verified by fluorescent microscopy. The fluorescent micrographsshowed spots of fluorescence associated with the cells as observed forthe PNA conjugates. In addition, a more diffuse fluorescence wasobserved throughout the cells with fluorescence observed in the nucleiof some cells.

[0192] When other cell lines were used (COS-7, green monkey kidneyderived cells; and NIH 3T3, mouse fibroblast cells) identical uptakepatterns were observed.

EXAMPLE 41 Cellular Uptake of an Adamantyl-Conjugated PNA.

[0193] The cellular uptake of an adamantyl-PNA (prepared according toExamples 35 or 36) was determined, and was also compared to the uptakeof a phosphorothioate oligonucleotide. The adamantyl-conjugated PNA andoligonucleotide were added directly to subconfluent HeLa cells at 1 μMconcentrations and left over night. The cells were next fixed and uptakevisualized by fluorescence microscopy. The oligonucleotide exhibitedfine punctate fluorescence, mainly confined to clusters in the cytoplasmof the cells and absent from the nuclei. With the PNA, punctatefluorescence was similarly observed. However, the spots were somewhatlarger and present both in the cytoplasm and on the cell membrane.

[0194] In an attempt to improve uptake of the adamantyl-PNA, the PNA wascombined with various commercially available cationic liposomes normallyused for transfection of DNA. In addition, PNA-containing liposomescomposed of the lipids DOPE and DDAB were also prepared. In theory, thehydrophobic adamantyl-group of the PNA should insert into the lipidlayer of the liposomes and thus entrap the PNA. The liposomes wereprepared by a simple ethanol injection technique, which was reported tobe efficient for the transport of plasmid DNA into cells. The differentPNA-liposome mixtures were fed to cells with a final PNA concentrationof 1 μM and incubated over night. The presence of either of the liposomereagents or the DOPE/DDAB liposomes resulted in a much more diffusefluorescence inside the cells, compared to when the PNA was added alone.However, the fluorescence was still confined to the cytoplasm with nosign of nuclear uptake. In contrast, when the oligonucleotide was mixedwith Lipofectamine™ and fed to the cells at a concentration of 1 μM, themajority of the cells had fluorescently stained nuclei.

[0195] In conclusion, adding adamantyl-conjugated PNA to cells resultedin an uptake pattern reminiscent of an endocytotic pathway, where thePNA ends up in endosomal or lysosomal compartments of the cell. When PNAis pre-mixed with liposome transfection reagents or incorporated intoDOPE/DDAB liposomes, it is distributed throughout the cell cytoplasm ina much more diffuse fashion.

[0196] Those skilled in the art will appreciate that numerous changesand modifications may be made to the preferred embodiments of thepresent invention and that such changes and modifications may be madewithout departing from the spirit of the invention. It is thereforeintended that the appended claims cover all such equivalent variationsas fall within the true spirit and scope of the invention.

What is claimed is:
 1. A peptide nucleic acid having formula:

wherein: each L is, independently, a naturally-occurring nucleobase or anon-naturally-occurring nucleobase; each R^(T) is hydrogen or the sidechain of a naturally-occurring or non-naturally-occurring amino acid, atleast one R^(T) being the side chain of a naturally-occurring ornon-naturally-occurring amino acid; R^(h) is OH, NH₂, or NHLysNH₂; eachof R^(i) and R^(j) is, independently, a lipophilic group or an aminoacid labeled with a fluorescent group; or R^(i) and R^(j), together, area lipophilic group; and n is an integer from 1 to
 30. 2. The peptidenucleic acid of claim 1 wherein at least one of said R^(T) is the sidechain of a naturally-occurring amino acid.
 3. The peptide nucleic acidof claim 2 wherein at least one R^(T) is the side chain of D-lysine. 4.The peptide nucleic acid of claim 1 wherein R^(i) is D-lysine labeledwith a fluorescent group and R^(j) is an adamantoyl group.
 5. Thepeptide nucleic acid of claim 4 wherein said fluorescent group isfluorescein.
 6. The peptide nucleic acid of claim 1 wherein R^(i) andR^(j), together, are an adamantoyl group.
 7. The peptide nucleic acid ofclaim 1 wherein R^(T) is the side chain of an amino acid and the carbonatom to which the side chain is attached is stereochemically enriched.8. A composition comprising a peptide nucleic acid incorporated into aliposome, said peptide nucleic acid having formula:

wherein: each L is, independently, a naturally-occurring nucleobase or anon-naturally-occurring nucleobase; each R^(T) is hydrogen or the sidechain of a naturally-occurring or non-naturally-occurring amino acid;R^(h) is OH, NH₂, or NHLysNH_(2;) each of R^(i) and R^(j) is,independently, a lipophilic group or an amino acid labeled with afluorescent group; or R^(i) and R^(j), together, are a lipophilic group;and n is an integer from 1 to
 30. 9. The composition of claim 8 whereinat least one of said R^(T) is the side chain of a naturally-occurringamino acid.
 10. The composition of claim 9 wherein said amino acid isD-lysine.
 11. The composition of claim 8 wherein R^(i) is D-lysinelabeled with a fluorescent group and R^(j) is an adamantoyl group. 12.The composition of claim 11 wherein said fluorescent group isfluorescein.
 13. The composition of claim 8 wherein R^(i) and R^(j),together, are an adamantoyl group.
 14. The composition of claim 8wherein R^(T) is the side chain of an amino acid and the carbon atom towhich the side chain is attached is stereochemically enriched.
 15. Amethod of modulating cellular uptake and distribution of a peptidenucleic acid comprising the steps of: (a) derivatizing a backboneposition of said peptide nucleic acid; and (b) conjugating thederivatized peptide nucleic acid of step (a) with a lipophilic group.16. The method of claim 15 wherein said derivatizing comprises attachingthe side chain of at least one naturally-occurring ornon-naturally-occurring amino acid to the backbone of said peptidenucleic acid.
 17. The method of claim 16 wherein said derivatizingcomprises attaching the side chain of a naturally-occurring amino acidto the backbone of said peptide nucleic acid.
 18. The method of claim 17wherein said amino acid is D-lysine.
 19. The method of claim 15 whereinsaid lipophilic group is an adamantyl group.
 20. The method of claim 15further comprising introducing the peptide nucleic acid of step (b) intoliposomes.
 21. A method of modulating cellular uptake and distributionof a peptide nucleic acid comprising the steps of: (a) conjugating saidpeptide nucleic acid with a lipophilic group; and (b) introducing theconjugated peptide nucleic acid of step (a) into liposomes.
 22. Themethod of claim 21 wherein said lipophilic group is an adamantyl group.23. A pharmaceutical composition comprising the peptide nucleic acidaccording to claim 1 and at least one pharmaceutically acceptablecarrier, binder, thickener, diluent, buffer, preservative or surfaceactive agent.
 24. A pharmaceutical composition comprising thecomposition of claim 8 and at least one pharmaceutically acceptablecarrier, binder, thickener, diluent, buffer, preservative or surfaceactive agent.
 25. A method of modulating cellular uptake anddistribution of a peptide nucleic acid in a cell or tissue comprisingadministering to the cell or tissue a peptide nucleic acid havingformula:

wherein: each L is, independently, a naturally-occurring nucleobase or anon-naturally-occurring nucleobase; each R^(T) is hydrogen or the sidechain of a naturally-occurring or non-naturally-occurring amino acid, atleast one R^(T) being the side chain of a naturally-occurring ornon-naturally-occurring amino acid; R^(h) is OH, NH₂, or NHLysNH_(2;)each of R^(i) and R^(j) is, independently, a lipophilic group or anamino acid labeled with a fluorescent group; or R^(i) and R^(j),together, are a lipophilic group; and n is an integer from 1 to
 30. 26.The method of claim 25 wherein at least one of said R^(T) is the sidechain of a naturally-occurring amino acid.
 27. The method of claim 26wherein said amino acid is D-lysine.
 28. The method of claim of claim 25wherein R^(i) is D-lysine labeled with a fluorescent group and R^(j) isan adamantoyl group.
 29. The method of claim 28 wherein said fluorescentgroup is fluorescein.
 30. The method of claim 25 wherein R^(i) andR^(j), together, are an adamantoyl group.
 31. The method of claim 25wherein R^(T) is the side chain of an amino acid and the carbon atom towhich the side chain is attached is stereochemically enriched.
 32. Amethod of modulating cellular uptake and distribution of a peptidenucleic acid in a cell or tissue comprising administering to the cell ortissue a composition comprising a peptide nucleic acid incorporated intoa liposome, said peptide nucleic acid having formula:

wherein: each L is, independently, a naturally-occurring nucleobase or anon-naturally-occurring nucleobase; each R^(T) is hydrogen or the sidechain of a naturally-occurring or non-naturally-occurring amino acid;R^(h) is OH, NH₂, or NHLysNH_(2;) each of R^(i) and R^(j) is,independently, a lipophilic group or an amino acid labeled with afluorescent group; or R^(i) and R^(j), together, are a lipophilic group;and n is an integer from 1 to
 30. 33. The method of claim 32 wherein atleast one of said R^(T) is the side chain of a naturally-occurring aminoacid.
 34. The method of claim 33 wherein said amino acid is D-lysine.35. The method of claim 32 wherein R^(i) is D-lysine labeled with afluorescent group and R^(j) is an adamantoyl group.
 36. The method ofclaim 35 wherein said fluorescent group is fluorescein.
 37. The methodof claim 32 wherein R^(i) and R^(j), together, are an adamantoyl group.38. The method of claim 32 wherein R^(T) is the side chain of an aminoacid and the carbon atom to which the side chain is attached isstereochemically enriched.
 39. A method of treating an animal comprisingadministering to the animal a therapeutically effective amount of apeptide nucleic acid of formula:

wherein: each L is, independently, a naturally-occurring nucleobase or anon-naturally-occurring nucleobase; each R^(T) is hydrogen or the sidechain of a naturally-occurring or non-naturally-occurring amino acid, atleast one R^(T) being the side chain of a naturally-occurring ornon-naturally-occurring amino acid; R^(h) is OH, NH₂, or NHLysNH_(2;)each of R^(i) and R^(j) is, independently, a lipophilic group or anamino acid labeled with a fluorescent group; or R^(i) and R^(j),together, are a lipophilic group; and n is an integer from 1 to
 30. 40.The method of claim 39 wherein at least one of said R^(T) is the sidechain of a naturally-occurring amino acid.
 41. The method of claim 40wherein at least one R^(T) is the side chain of D-lysine.
 42. The methodof claim 39 wherein R^(i) is D-lysine labeled with a fluorescent groupand R^(j) is an adamantoyl group.
 43. The method of claim 42 whereinsaid fluorescent group is fluorescein.
 44. The method of claim 39wherein R^(i) and R^(j), together, are an adamantoyl group.
 45. Themethod of claim 39 wherein R^(T) is the side chain of an amino acid andthe carbon atom to which the side chain is attached is stereochemicallyenriched.
 46. A method of treating an animal comprising administering tothe animal a therapeutically effective amount of a compositioncomprising a peptide nucleic acid incorporated into a liposome, saidpeptide nucleic acid having formula:

wherein: each L is, independently, a naturally-occurring nucleobase or anon-naturally-occurring nucleobase; each R^(T) is hydrogen or the sidechain of a naturally-occurring or non-naturally-occurring amino acid;R^(h) is OH, NH₂, or NHLysNH_(2;) each of R^(i) and R^(j) is,independently, a lipophilic group or an amino acid labeled with afluorescent group; or R^(i) and R^(j), together, are a lipophilic group;and n is an integer from 1 to
 30. 47. The method of claim 46 wherein atleast one of said R^(T) is the side chain of a naturally-occurring aminoacid.
 48. The method of claim 47 wherein said amino acid is D-lysine.49. The method of claim 46 wherein R^(i) is D-lysine labeled with afluorescent group and R^(j) is an adamantoyl group.
 50. The method ofclaim 46 wherein said fluorescent group is fluorescein.
 51. The methodof claim 46 wherein R^(i) and R^(j), together, are an adamantoyl group.52. The method of claim 46 wherein R^(T) is the side chain of an aminoacid and the carbon atom to which the side chain is attached isstereochemically enriched.